LOCUS       sequence-193530-        7881 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAVS1-targeting expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7881)
  AUTHORS   Chen X, Janssen JM, Liu J, Maggio I, 't Jong AEJ, Mikkers HMM, 
            Goncalves MAFV
  TITLE     In trans paired nicking triggers seamless genome editing without 
            double-stranded DNA cutting.
  JOURNAL   Nat Commun. 2017 Sep 22;8(1):657. doi: 10.1038/s41467-017-00687-1.
  PUBMED    28939824
REFERENCE   2  (bases 1 to 7881)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2017-09-22"; volume: "8(1):657. doi"; pages: " 
            10.1038/s41467-017-00687-1"
COMMENT     Sequence Label: AV11_pDonor.EP.S1
FEATURES             Location/Qualifiers
     source          1..7881
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    926..1729
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        1740..2249
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2169..2187
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2272..2868
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2272..2291)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2768..2788
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2878..2931
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      2938..2947
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2944..2946,2947..2949,2950..3663)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2944..2946,2947..2949,2950..3660)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2989..3010)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3250..3269)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3597..3618
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3690..3707)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3696..3920
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3928..4764
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(5557..5576)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5558..5576)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5583..5599)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5666..5683)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5837..6425)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5917..5936)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6596..7387,7388..7456))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7219..7238
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7457..7561)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7629..7647
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tccaccgcgg
      181 tggcggcccg ccgagctcga ccccgccgcg ccgccgcccg cccgcgccgt gctggactcc
      241 accaacgccg acggtatcag cgccctgcac caggtcagcg ccccccgccc ggcgtctccc
      301 ggggccaggt ccaccctctg ctgcgccacc tggggcatcc tccttccccg ttgccagtct
      361 cgatccgccc cgtcgttcct ggccctgggc tttgccaccc tatgctgaca ccccgtccca
      421 gtccccctta ccattcccct tcgaccaccc cacttccgaa ttggagccgc ttcaactggc
      481 cctgggctta gccactctgt gctgaccact ctgccccagg cctccttacc attccccttc
      541 gacctactct cttccgcatt ggagtcgctt taactggccc tggctttggc agcctgtgct
      601 gacccatgca gtcctcctta ccatccctcc ctcgacttcc cctcttccga tgttgagccc
      661 ctccagccgg tcctggactt tgtctccttc cctgccctgc cctctcctga acctgagcca
      721 gctcccatag ctcagtctgg tctatctgcc tggccctggc cattgtcact ttgcgctgcc
      781 ctcctctcgc ccccgagtgc ccttgctgtg ccgccggaac tctgccctct aacgctgccg
      841 tctctctcct gagtccggac cactttgagc tctactggct tctgcgccgc ctctggccca
      901 ctgtttcccc ttcccaggca ggtcctgctt tctctgacct gcattctctc ccctgggcct
      961 gtgccgcttt ctgtctgcag cttgtggcct gggtcacctc tacggctggc ccagatcctt
     1021 ccctgccgcc tccttcaggt tccgtcttcc tccactccct cttccccttg ctctctgctg
     1081 tgttgctgcc caaggatgct ctttccggag cacttccttc tcggcgctgc accacgtgat
     1141 gtcctctgag cggatcctcc ccgtgtctgg gtcctctccg ggcatctctc ctccctcacc
     1201 caaccccatg ccgtcttcac tcgctgggtt cccttttcct tctccttctg gggcctgtgc
     1261 catctctcgt ttcttaggat ggccttctcc gacggatgtc tcccttgcgt cccgcctccc
     1321 cttcttgtag gcctgcatca tcaccgtttt tctggacaac cccaaagtac cccgtctccc
     1381 tggctttagc cacctctcca tcctcttgct ttctttgcct ggacaccccg ttctcctgtg
     1441 gattcgggtc acctctcact cctttcattt gggcagctcc cctacccccc ttacctctct
     1501 agtctgtgct agctcttcca gccccctgtc atggcatctt ccaggggtcc gagagctcag
     1561 ctagtcttct tcctccaacc cgggccccta tgtccacttc aggacagcat gtttgctgcc
     1621 tccagggatc ctgtgtcccc gagctgggac caccttatat tcccagggcc ggttaatgtg
     1681 gctctggttc tgggtacttt tatctgtccc ctccacccca cagtggggca agcttccacg
     1741 gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct
     1801 gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc
     1861 gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct
     1921 gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg
     1981 gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg
     2041 cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc
     2101 ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg
     2161 cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc
     2221 gttgaccgaa tcaccgacct ctctccccac cgggtacgtc gctagcccac catgaccgag
     2281 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt acgcaccctc
     2341 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgacccgga ccgccacatc
     2401 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     2461 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     2521 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     2581 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     2641 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     2701 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     2761 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     2821 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg atcgggagag
     2881 ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc accggtcgcc
     2941 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     3001 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     3061 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     3121 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     3181 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     3241 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     3301 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     3361 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     3421 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     3481 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     3541 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     3601 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3661 taaagcggcc gcgtcgagtc taggatcagc ctcgactgtg ccttctagtt gccagccatc
     3721 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3781 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3841 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3901 ggatgcggtg ggctctatgg aagctttact agggacagga ttggtgacag aaaagcccca
     3961 tccttaggcc tcctccttcc tagtctcctg atattgggtc taacccccac ctcctgttag
     4021 gcagattcct tatctggtga cacaccccca tttcctggag ccatctctct ccttgccaga
     4081 acctctaagg tttgcttacg atggagccag agaggatcct gggagggaga gcttggcagg
     4141 gggtgggagg gaaggggggg atgcgtgacc tgcccggttc tcagtggcca ccctgcgcta
     4201 ccctctccca gaacctgagc tgctctgacg cggctgtctg gtgcgtttca ctgatcctgg
     4261 tgctgcagct tccttacact tcccaagagg agaagcagtt tggaaaaaca aaatcagaat
     4321 aagttggtcc tgagttctaa ctttggctct tcacctttct agtccccaat ttatattgtt
     4381 cctccgtgcg tcagttttac ctgtgagata aggccagtag ccagccccgt cctggcaggg
     4441 ctgtggtgag gaggggggtg tccgtgtgga aaactccctt tgtgagaatg gtgcgtccta
     4501 ggtgttcacc aggtcgtggc cgcctctact ccctttctct ttctccatcc ttctttcctt
     4561 aaagagtccc cagtgctatc tgggacatat tcctccgccc agagcagggt cccgcttccc
     4621 taaggccctg ctctgggctt ctgggtttga gtccttggca agcccaggag aggcgctcag
     4681 gcttccctgt cccccttcct cgtccaccat ctcatgcccc tggctctcct gccccttccc
     4741 tacaggggtt cctggctctg ctcttcagac tgagccccgt tcccctgcat ccccgttccc
     4801 ctgcatcccc cttcccctgc atcccccaga ggccccaggc cacctacttg gcctggaccc
     4861 cacgagaggc caccccagcc ctgtctacca ggctgccttt tgggtggatt ctcctccaac
     4921 tgtggggtga ctgcttggca aactcactct tcggggtatc ccaggaggcc tggagcattg
     4981 gggtgggctg gggttcagag aggagggatt cccttctcag gttacgtggc caagaagcag
     5041 gggagctggg tttgggtcag gtctgggtgt ggggtgacca gcttatgctg tttgcccagg
     5101 acagcctagt tttagcactg aaaccctcag tcctaggtgt tcaccaggtc gtggccgcct
     5161 ctactccctt tctctttctc catccttctt tccttaaaga gtccccagtg ctatctggga
     5221 catattcctc cgcccagagc agggtcccgc ttccctaagg ccctgctctg ggcttctggg
     5281 tttgagtcct tggcaagccc aggagaggcg ctcaggcttc cctgtccccc ttcctcgtcc
     5341 accatctcat gcccctggct ctcctgcccc ttccctacag gggttcctgg ctctgctcta
     5401 agggcgaatt cgatctggcg ccatcgatac gcgtacgtcg cgaccgcgga catgtacaga
     5461 gctcgagaag tactagtggc cacgtgggcc gtgcacctta agcttttaaa taaggaggaa
     5521 taacatatga ccatgattac gccaagctcc aattcgccct atagtgagtc gtattacaat
     5581 tcactggccg tcgttttact atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     5641 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5701 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5761 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5821 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5881 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5941 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6001 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6061 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6121 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6181 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6241 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     6301 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6361 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6421 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6481 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6541 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6601 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6661 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6721 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6781 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6841 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6901 ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6961 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7021 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7081 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7141 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7201 gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7261 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7321 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7381 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7441 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7501 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7561 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     7621 cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattggaa acagctatga
     7681 ccatgattac gccaagctcg aaattacccc tcactaaagg gaacaaagct ggtacgagga
     7741 caggctggag ccatgggcat ggctactcaa gctgatttga tggagttgga catggccatg
     7801 gctggtgacc acgtcgtgga atgccttcga attcagcacc tgcacatggg acgtcgacct
     7861 gaggtaatta gtaccgcggc c
//
