LOCUS       pLVXS-EF1a-Cas9-PGK-Puro Vector               11375 bp ds-DNA     circular SYN 01-SEP-2022
DEFINITION  synthetic circular DNA
COMMENT 	Created by Takara Bio USA, Inc. http://www.takarabio.com
COMMENT		Cat. No. 632646
FEATURES             Location/Qualifiers
     source          1..11375
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     LTR             1..635
                     /label=5' LTR
     misc_feature    681..806
                     /label="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_binding    1303..1536
                     /label=RRE
		     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the cytoplasm."
     misc_feature    2028..2143
                     /label="cPPT/CTS"
		     /note="central polypurine tract"
     promoter        2209..2420
                     /label="EF1a"
		     /note="EF1-alpha promoter"
     CDS             2501..2521
                     /label="SV40 NLS"
		     /note="nuclear localization signal of SV40 large T 
                     antigen"
     misc_feature    2546..6646
                     /label="hCas9"
		     /note="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             6659..6679
                     /label="SV40 NLS"
                     /note="nuclear localization signal of SV40 large T 
                     antigen"
     promoter        6699..7198
                     /label="PGK Promoter"
		     /note="phosphoglycerate kinase promoter"
     CDS             7219..7818
                     /label="Puro(R)"
                     /note="Puromycin resistance gene"
     misc_feature    7832..8420
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional regulatory element"
     LTR             8632..8865
                     /label="3' SIN LTR"
		     /note="3’ self-inactivating long terminal repeat"
     rep_origin      complement(9302..9890)
                     /direction=LEFT
                     /label=pUC origin
		     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
     CDS             complement(10061..10921)
                     /label="Amp(R)"
		     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag
     2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2161 acagcagaga tccagtttat cgatcgtgag gctccgatgc ccgtcagtgg gcagagcgca
     2221 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
     2281 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     2341 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     2401 ggtttgccgc cagaacacag gatccatgga ctataaggac cacgacggag actacaagga
     2461 tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga agcggaaggt
     2521 cggtatccac ggagtcccag cagccgacaa gaagtactcc attgggctcg atatcggcac
     2581 aaacagcgtc ggctgggccg tcattacgga cgactacaag gtgccgagca aaaaattcaa
     2641 agttctgggc aataccgatc gccacagcat aaagaagaac ctcattggcg ccctcctgtt
     2701 cgactccggg gagacggccg aagccacgcg gctcaaaaga acagcacggc gcagatatac
     2761 ccgcagaaag aatcggatct gctacctgca ggagatcttt agtaatgaga tggctaaggt
     2821 ggatgactct ttcttccata ggctggagga gtcctttttg gtggaggagg ataaaaagca
     2881 cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg gcgtaccatg aaaagtaccc
     2941 aaccatatat catctgagga agaagcttgt agacagtact gataaggctg acttgcggtt
     3001 gatctatctc gcgctggcgc atatgatcaa atttcgggga cacttcctca tcgaggggga
     3061 cctgaaccca gacaacagcg atgtcgacaa actctttatc caactggttc agacttacaa
     3121 tcagcttttc gaagagaacc cgatcaacgc atccggagtt gacgccaaag caatcctgag
     3181 cgctaggctg tccaaatccc ggcggctcga aaacctcatc gcacagctcc ctggggagaa
     3241 gaagaacggc ctgtttggta atcttatcgc cctgtcactc gggctgaccc ccaactttaa
     3301 atctaacttc gacctggccg aagatgccaa gcttcaactg agcaaagaca cctacgatga
     3361 tgatctcgac aatctgctgg cccagatcgg cgaccagtac gcagaccttt ttttggcggc
     3421 aaagaacctg tcagacgcca ttctgctgag tgatattctg cgagtgaaca cggagatcac
     3481 caaagctccg ctgagcgcta gtatgatcaa gcgctatgat gagcaccacc aagacttgac
     3541 tttgctgaag gcccttgtca gacagcaact gcctgagaag tacaaggaaa ttttcttcga
     3601 tcagtctaaa aatggctacg ccggatacat tgacggcgga gcaagccagg aggaatttta
     3661 caaatttatt aagcccatct tggaaaaaat ggacggcacc gaggagctgc tggtaaagct
     3721 taacagagaa gatctgttgc gcaaacagcg cactttcgac aatggaagca tcccccacca
     3781 gattcacctg ggcgaactgc acgctatcct caggcggcaa gaggatttct accccttttt
     3841 gaaagataac agggaaaaga ttgagaaaat cctcacattt cggataccct actatgtagg
     3901 ccccctcgcc cggggaaatt ccagattcgc gtggatgact cgcaaatcag aagagaccat
     3961 cactccctgg aacttcgagg aagtcgtgga taagggggcc tctgcccagt ccttcatcga
     4021 aaggatgact aactttgata aaaatctgcc taacgaaaag gtgcttccta aacactctct
     4081 gctgtacgag tacttcacag tttataacga gctcaccaag gtcaaatacg tcacagaagg
     4141 gatgagaaag ccagcattcc tgtctggaga gcagaagaaa gctatcgtgg acctcctctt
     4201 caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa gactatttca aaaagattga
     4261 atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc ttcaacgcat ccctgggaac
     4321 gtatcacgat ctcctgaaaa tcattaaaga caaggacttc ctggacaatg aggagaacga
     4381 ggacattctt gaggacattg tcctcaccct tacgttgttt gaagataggg agatgattga
     4441 agaacgcttg aaaacttacg ctcatctctt cgacgacaaa gtcatgaaac agctcaagag
     4501 gcgccgatat acaggatggg ggcggctgtc aagaaaactg atcaatggga tccgagacaa
     4561 gcagagtgga aagacaatcc tggattttct taagtccgat ggatttgcca accggaactt
     4621 catgcagttg atccatgatg actctctcac ctttaaggag gacatccaga aagcacaagt
     4681 ttctggccag ggggacagtc ttcacgagca catcgctaat cttgcaggta gcccagctat
     4741 caaaaaggga atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag
     4801 gcataagccc gagaatatcg ttatcgagat ggcccgagag aaccaaacta cccagaaggg
     4861 acagaagaac agtagggaaa ggatgaagag gattgaagag ggtataaaag aactggggtc
     4921 ccaaatcctt aaggaacacc cagttgaaaa cacccagctt cagaatgaga agctctacct
     4981 gtactacctg cagaacggca gggacatgta cgtggatcag gaactggaca tcaatcggct
     5041 ctccgactac gacgtggatc atatcgtgcc ccagtctttt ctcaaagatg attctattga
     5101 taataaagtg ttgacaagat ccgataaaaa tagagggaag agtgataacg tcccctcaga
     5161 agaagttgtc aagaaaatga aaaattattg gcggcagctg ctgaacgcca aactgatcac
     5221 acaacggaag ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg agttggataa
     5281 agccggcttc atcaaaaggc agcttgttga gacacgccag atcaccaagc acgtggccca
     5341 aattctcgat tcacgcatga acaccaagta cgatgaaaat gacaaactga ttcgagaggt
     5401 gaaagttatt actctgaagt ctaagctggt ctcagatttc agaaaggact ttcagtttta
     5461 taaggtgaga gagatcaaca attaccacca tgcgcatgat gcctacctga atgcagtggt
     5521 aggcactgca cttatcaaaa aatatcccaa gcttgaatct gaatttgttt acggagacta
     5581 taaagtgtac gatgttagga aaatgatcgc aaagtctgag caggaaatag gcaaggccac
     5641 cgctaagtac ttcttttaca gcaatattat gaattttttc aagaccgaga ttacactggc
     5701 caatggagag attcggaagc gaccacttat cgaaacaaac ggagaaacag gagaaatcgt
     5761 gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa
     5821 catcgttaaa aagaccgaag tacagaccgg aggcttctcc aaggaaagta tcctcccgaa
     5881 aaggaacagc gacaagctga tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg
     5941 attcgattct cctacagtcg cttacagtgt actggttgtg gccaaagtgg agaaagggaa
     6001 gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg agcgatcaag
     6061 cttcgaaaaa aaccccatcg actttctcga ggcgaaagga tataaagagg tcaaaaaaga
     6121 cctcatcatt aagcttccca agtactctct ctttgagctt gaaaacggcc ggaaacgaat
     6181 gctcgctagt gcgggcgagc tgcagaaagg taacgagctg gcactgccct ctaaatacgt
     6241 taatttcttg tatctggcca gccactatga aaagctcaaa gggtctcccg aagataatga
     6301 gcagaagcag ctgttcgtgg aacaacacaa acactacctt gatgagatca tcgagcaaat
     6361 aagcgaattc tccaaaagag tgatcctcgc cgacgctaac ctcgataagg tgctttctgc
     6421 ttacaataag cacagggata agcccatcag ggagcaggca gaaaacatta tccacttgtt
     6481 tactctgacc aacttgggcg cgcctgcagc cttcaagtac ttcgacacca ccatagacag
     6541 aaagcggtac acctctacaa aggaggtcct ggacgccaca ctgattcatc agtcaattac
     6601 ggggctctat gaaacaagaa tcgacctctc tcagctcggt ggagacagca gggctgaccc
     6661 caagaagaag aggaaggtgt aatctagata attctaccgg gtaggggagg cgcttttccc
     6721 aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg
     6781 cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg
     6841 tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca
     6901 gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat
     6961 ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct
     7021 ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag
     7081 gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac
     7141 gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac
     7201 ctgcagccca agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     7261 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     7321 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     7381 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     7441 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     7501 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     7561 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     7621 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     7681 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     7741 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     7801 cgcaagcccg gtgcctgacc gcgtctggaa caatcaacct ctggattaca aaatttgtga
     7861 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     7921 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     7981 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     8041 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     8101 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     8161 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     8221 ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     8281 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     8341 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     8401 cctttgggcc gcctccccgc ctggaattaa ttctgcagtc gagacctaga aaaacatgga
     8461 gcaatcacaa gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa
     8521 gaggaggagg aggtgggttt tccagtcaca cctcacctca ggtaccttta agaccaatga
     8581 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagagggga ctggaagggc
     8641 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
     8701 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     8761 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     8821 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc
     8881 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggcctt
     8941 gacattgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     9001 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     9061 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     9121 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     9181 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     9241 ccatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     9301 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     9361 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     9421 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     9481 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     9541 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     9601 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     9661 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     9721 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     9781 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     9841 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     9901 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     9961 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
    10021 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
    10081 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
    10141 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
    10201 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
    10261 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
    10321 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
    10381 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
    10441 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
    10501 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
    10561 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
    10621 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
    10681 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
    10741 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
    10801 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
    10861 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
    10921 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
    10981 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
    11041 aagtgccacc tgacgtcgac ggatcgggag atcaacttgt ttattgcagc ttataatggt
    11101 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
    11161 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcaa ctggataact
    11221 caagctaacc aaaatcatcc caaacttccc accccatacc ctattaccac tgccaattac
    11281 ctgtggtttc atttactcta aacctgtgat tcctctgaat tattttcatt ttaaagaaat
    11341 tgtatttgtt aaatatgtac tacaaactta gtagt
//
