LOCUS       sequence-193531-        8024 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 8024)
  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 8024)
  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: AV09_pDonor.EP.S1.TS
FEATURES             Location/Qualifiers
     source          1..8024
                     /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(3694..3711)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3700..3924
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3932..4768
                     /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(5593..5612)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5594..5612)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5619..5635)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5702..5719)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5873..6461)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5953..5972)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6632..7423,7424..7492))
                     /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     7255..7274
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     7771..7789
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7844..7862
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag ctcgaaatta 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 ggccgcgtcg agtctaggat cagcctcgac tgtgccttct agttgccagc
     3721 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3781 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3841 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3901 ctggggatgc ggtgggctct atggaagctt tactagggac aggattggtg acagaaaagc
     3961 cccatcctta ggcctcctcc ttcctagtct cctgatattg ggtctaaccc ccacctcctg
     4021 ttaggcagat tccttatctg gtgacacacc cccatttcct ggagccatct ctctccttgc
     4081 cagaacctct aaggtttgct tacgatggag ccagagagga tcctgggagg gagagcttgg
     4141 cagggggtgg gagggaaggg ggggatgcgt gacctgcccg gttctcagtg gccaccctgc
     4201 gctaccctct cccagaacct gagctgctct gacgcggctg tctggtgcgt ttcactgatc
     4261 ctggtgctgc agcttcctta cacttcccaa gaggagaagc agtttggaaa aacaaaatca
     4321 gaataagttg gtcctgagtt ctaactttgg ctcttcacct ttctagtccc caatttatat
     4381 tgttcctccg tgcgtcagtt ttacctgtga gataaggcca gtagccagcc ccgtcctggc
     4441 agggctgtgg tgaggagggg ggtgtccgtg tggaaaactc cctttgtgag aatggtgcgt
     4501 cctaggtgtt caccaggtcg tggccgcctc tactcccttt ctctttctcc atccttcttt
     4561 ccttaaagag tccccagtgc tatctgggac atattcctcc gcccagagca gggtcccgct
     4621 tccctaaggc cctgctctgg gcttctgggt ttgagtcctt ggcaagccca ggagaggcgc
     4681 tcaggcttcc ctgtccccct tcctcgtcca ccatctcatg cccctggctc tcctgcccct
     4741 tccctacagg ggttcctggc tctgctcttc agactgagcc ccgttcccct gcatccccct
     4801 tcccctgcat cccccagagg ccccaggcca cctacttggc ctggacccca cgagaggcca
     4861 ccccagccct gtctaccagg ctgccttttg ggtggattct cctccaactg tggggtgact
     4921 gcttggcaaa ctcactcttc ggggtatccc aggaggcctg gagcattggg gtgggctggg
     4981 gttcagagag gagggattcc cttctcaggt tacgtggcca agaagcaggg gagctgggtt
     5041 tgggtcaggt ctgggtgtgg ggtgaccagc ttatgctgtt tgcccaggac agcctagttt
     5101 tagcgctgaa accctcagtc ctaggtgttc accaggtcgt ggccgcctct actccctttc
     5161 tctttctcca tccttctttc cttaaagagt ccccagtgct atctgggaca tattcctccg
     5221 cccagagcag ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt tgagtccttg
     5281 gcaagcccag gagaggcgct caggcttccc tgtccccctt cctcgtccac catctcatgc
     5341 ccctggctct cctgcccctt ccctacaggg gttcctggct ctgctctaag ggcgaattcg
     5401 atatcaagct tatcgatacc gtcgacacgc gtctgtcccc tccaccccac agtggggcca
     5461 ctagggacag gattggtgac agactcgact cgaagatctc gagaagtact agtggccacg
     5521 tgggccgtgc accttaagct tttaaataag gaggaataac atatgaccat gattacgcca
     5581 agctccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt tttactatgc
     5641 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     5701 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5761 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5821 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5881 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5941 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6001 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6061 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6121 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6181 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6241 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6301 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6361 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6421 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6481 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6541 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6601 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6661 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6721 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6781 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6841 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6901 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg
     6961 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7021 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7081 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7141 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7201 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata
     7261 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7321 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7381 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7441 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7501 tttttcaaaa ttcgatatca agcttatcga taccgtcgac acgcgtctgt cccctccacc
     7561 ccacagtggg gccactaggg acaggattgg tgacagactc gactcgaaga tctcgattat
     7621 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7681 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     7741 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt
     7801 caagaattgg aaacagctat gaccatgatt acgccaagct cgaaattaac cctcactaaa
     7861 gggaacaaaa gctggtacga ggacaggctg gagccatggg catggctact caagctgatt
     7921 tgatggagtt ggacatggcc atggctggtg accacgtcgt ggaatgcctt cgaattcagc
     7981 acctgcacat gggacgtcga cctgaggtaa ttagtaccgc ggcc
//
