LOCUS       Exported                7070 bp ds-DNA     circular SYN 27-NOV-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pAAV2_SauriCas9_U6_BsaI
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7070)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Nov 28, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7070
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..130
                     /label=AAV2 ITR (alternate）
     misc_feature    1..130
                     /label=ITR
     enhancer        154..533
                     /label=CMV enhancer
     promoter        534..737
                     /label=CMV promoter
     CDS             768..788
                     /codon_start=1
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             813..2960
                     /codon_start=1
                     /label=Cas12J-8
                     /translation="IKPTVSQFLTPGFKLIRNHSRTAGLKLKNEGEEACKKFVRENEIP
                     KDECPNFQGGPAIANIIAKSREFTEWEIYQSSLAIQEVIFTLPKDKLPEPILKEEWRAQ
                     WLSEHGLDTVPYKEAAGLNLIIKNAVNTYKGVQVKVDNKNKNNLAKINRKNEIAKLNGE
                     QEISFEEIKAFDDKGYLLQKPSPNKSIYCYQSVSPKPFITSKYHNVNLPEEYIGYYRKS
                     NEPIVSPYQFDRLRIPIGEPGYVPKWQYTFLSKKENKRRKLSKRIKNVSPILGIICIKK
                     DWCVFDMRGLLRTNHWKKYHKPTDSINDLFDYFTGDPVIDTKANVVRFRYKMENGIVNY
                     KPVREKKGKELLENICDQNGSCKLATVDVGQNNPVAIGLFELKKVNGELTKTLISRHPT
                     PIDFCNKITAYRERYDKLESSIKLDAIKQLTSEQKIEVDNYNNNFTPQNTKQIVCSKLN
                     INPNDLPWDKMISGTHFISEKAQVSNKSEIYFTSTDKGKTKDVMKSDYKWFQDYKPKLS
                     KEVRDALSDIEWRLRRESLEFNKLSKSREQDARQLANWISSMCDVIGIENLVKKNNFFG
                     GSGKREPGWDNFYKPKKENRWWINAIHKALTELSQNKGKRVILLPAMRTSITCPKCKYC
                     DSKNRNGEKFNCLKCGIELNADIDVATENLATVAITAQSMPKPTCERSGDAKKPVRARK
                     AKAPEFHDKLAPSYTVVLREAV"
     CDS             2961..3008
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     misc_feature    2961..3008
                     /label=Deletion N2
     CDS             3030..3086
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3087..3683
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     CDS             3684..3710
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3711..3737
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3738..3764
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3798..4005
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4013..4261
                     /label=U6 Promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    4263..4298
                     /label=Cas12j-8-repeat
     misc_feature    4299..4317
                     /label=gRNA
     repeat_region   4333..4473
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4548..5003
                     /direction=RIGHT
                     /label=f1 ori
     promoter        5285..5389
                     /label=AmpR promoter
     CDS             5390..6250
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6421..7009
                     /direction=RIGHT
                     /label=ori
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc
      781 ggaaggtcgg tatccacgga gtcccagcag ccatcaagcc aaccgtctcc cagttcctga
      841 cacctggctt taagctgatc agaaaccaca gcaggacagc cggcctgaag ctgaagaatg
      901 agggagaaga agcttgtaaa aaattcgtgc gggagaacga gatccctaaa gacgagtgcc
      961 ccaacttcca gggcggccct gccatcgcta acatcatcgc caagagcaga gaatttaccg
     1021 agtgggagat ctaccagtct agcctggcta ttcaggaggt gatcttcacc ctgcctaaag
     1081 ataagctgcc tgaacctatc ctgaaggaag aatggagagc ccaatggctg tctgagcacg
     1141 gcctggacac cgttccttac aaggaggccg ccggactcaa cctgatcatc aagaacgccg
     1201 tgaataccta caagggcgtg caggtgaagg tggacaacaa gaacaagaac aacctggcca
     1261 agatcaatag aaagaatgag atcgccaagc tgaacggaga gcaggagatc agcttcgagg
     1321 aaatcaaggc ctttgacgac aagggatacc tgctgcagaa gcccagccct aacaagtcca
     1381 tctattgcta ccaatctgtg tccccaaagc ccttcatcac cagcaaatac cacaacgtga
     1441 acctgccaga ggagtacatc ggctactaca gaaagtctaa tgaacccatc gtgtctccct
     1501 accagttcga tagactgcgg atccccatcg gcgagcctgg atatgtgcct aagtggcagt
     1561 acaccttcct gtctaagaag gaaaacaaac ggagaaagct ttctaaaagg atcaagaatg
     1621 taagccccat cctgggcatc atttgtatca aaaaggactg gtgcgtgttc gacatgcggg
     1681 gactgctgag aaccaaccat tggaagaagt accacaagcc tacagatagc atcaatgatc
     1741 tgttcgacta ttttacaggc gaccccgtga tcgacacaaa ggccaatgtg gtcagattcc
     1801 ggtacaagat ggaaaatggc atcgtgaatt acaaacccgt gcgcgaaaaa aagggcaaag
     1861 agctgctgga aaacatttgc gatcaaaacg gtagctgcaa gctggctaca gtcgatgtgg
     1921 gccagaacaa ccccgtggcc atcggcctct tcgagctgaa gaaggtgaac ggcgagctga
     1981 ccaagacact gatcagcaga cacccaaccc ctatcgactt ctgcaacaag atcaccgcct
     2041 accgggaaag atacgacaag ctggaaagct ctattaagct ggacgccatc aagcagctga
     2101 ccagcgagca gaaaatcgag gtggacaact acaacaacaa cttcacccct cagaacacca
     2161 agcagatcgt gtgcagcaag ctgaacatca accccaacga cctgccttgg gacaagatga
     2221 tcagcggcac acacttcatc tctgaaaagg cacaggtgag caacaagtct gagatctact
     2281 tcacctccac cgacaagggc aagacaaagg acgtgatgaa gagcgactac aagtggtttc
     2341 aagactacaa gcctaagctg tctaaggaag tgcgagacgc cctgtcagat atcgagtggc
     2401 ggctgagaag agagagcctg gaattcaaca aactgagcaa aagcagagag caggacgcca
     2461 gacagctggc caactggatc agcagcatgt gcgatgtgat cggcatcgag aacctggtga
     2521 aaaagaacaa cttcttcggc ggcagcggca aacgggaacc tggctgggat aatttctaca
     2581 agcctaagaa ggaaaaccgg tggtggatca acgccatcca caaggctctg actgaactgt
     2641 cccagaacaa gggcaagaga gtgatcttac tgcctgctat gcggaccagc atcacctgtc
     2701 ctaagtgtaa atactgtgat agcaagaaca gaaacggcga gaagttcaac tgcctgaagt
     2761 gcggcatcga gctgaacgcc gacattgatg ttgctacaga gaatctggcc accgtggcca
     2821 ttaccgcgca gtccatgcct aagcccacct gcgagagaag cggcgatgcc aagaaaccgg
     2881 tccgcgccag aaaggccaaa gcccctgagt ttcacgacaa gctcgctcct agctacacag
     2941 tcgtgctgag agaggccgtg aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa
     3001 agaaaaaggg atccgctagc ggcagcggcg ccaccaactt cagcctgctg aagcaggccg
     3061 gcgacgtgga ggagaacccc ggccccatga ccgagtacaa gcccacggtg cgcctcgcca
     3121 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     3181 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     3241 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     3301 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     3361 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     3421 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc
     3481 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     3541 agcgcgccgg ggtgcccgcc ttcctggaaa cctccgcgcc ccgcaacctc cccttctacg
     3601 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     3661 gcatgacccg caagcccggt gcctacccat acgatgttcc agattacgct tacccatacg
     3721 atgttccaga ttacgcttac ccatacgatg ttccagatta cgcttaagaa ttcctagagc
     3781 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3841 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3901 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3961 cagcaagggg gaggattggg aagagaatag caggcatgct ggggaggtac ctgagggcct
     4021 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
     4081 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     4141 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     4201 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
     4261 cgctttcaag actaatagat tgctccttac gaggagacca gtcttctcag aagacgtttt
     4321 ttttgcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     4381 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     4441 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     4501 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     4561 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4621 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4681 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4741 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     4801 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4861 aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc
     4921 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4981 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     5041 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     5101 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5161 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     5221 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     5281 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5341 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5401 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5461 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5521 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     5581 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5641 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5701 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5761 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     5821 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     5881 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     5941 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     6001 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     6061 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat
     6121 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6181 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6241 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     6301 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6361 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6421 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6481 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6541 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     6601 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6661 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6721 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6781 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6841 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6901 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6961 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     7021 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
