LOCUS       sequence-396443-        5937 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid for targeting dTAG into the AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5937)
  AUTHORS   van der Woude M, Davo-Martinez C, Thijssen KL, Vermeulen W, Lans H
  TITLE     Recovery of protein synthesis to assay DNA repair activity in 
            transcribed genes in living cells and tissues.
  JOURNAL   Nucleic Acids Res. 2023 Oct 13;51(18):e93. doi: 10.1093/nar/gkad642.
  PUBMED    37522336
REFERENCE   2  (bases 1 to 5937)
  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: "Nucleic 
            Acids Res."; date: "2023-10-13"; volume: "51(18):e93. doi"; pages: "
            10.1093/nar/gkad642"
COMMENT     Sequence Label: PGK-EGFP-FKBPF36V
FEATURES             Location/Qualifiers
     source          1..5937
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(20..38)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        106..210
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(211..279,280..1071)
                     /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"
     primer_bind     complement(429..448)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1242..1830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1731..1750
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1984..2001
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2118..2139
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2154..2159,2160..2177,2178..2184)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2192..2208
                     /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     2197..2219
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2216..2232
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2216..2232
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2525..3025
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2944..2962
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      3041..3050
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3047..3049,3050..3052,3053..3763)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3047..3049,3050..3052,3053..3763)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3092..3113)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3353..3372)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3700..3721
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3815..4135
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             4139..4159
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4286..4407
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4323..4342)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4377..4396
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4438..4649
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      4668..4677
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4674..5072
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(5260..5279)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     primer_bind     complement(5553..5570)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5553..5569)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5562..5584)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5778..5797)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5897..5919
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
       61 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
      121 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
      181 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
      241 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
      301 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
      361 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
      421 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
      481 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
      541 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
      601 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
      661 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
      721 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
      781 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
      841 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
      901 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
      961 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     1021 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     1081 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     1141 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     1201 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1261 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctgg tagcggtggt ttttttgccg
     1321 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1381 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1441 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1501 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1561 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1621 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1681 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1741 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     1801 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     1861 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     1921 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     1981 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     2041 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     2101 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     2161 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     2221 aacagctatg accatgatta cgccaagctt gcatgcaggc ctctgcagca tggcatcttc
     2281 caggggtccg agagctcagc tagtcttctt cctccaaccc gggcccctat gtccacttca
     2341 ggacagcatg tttgctgcct ccagggatcc tgtgtccccg agctgggacc accttatatt
     2401 cccagggccg gttaatgtgg ctctggttct gggtactttt atctgtcccc tccaccccac
     2461 agtggggcaa ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     2521 tccgttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct gctctgggcg
     2581 tggttccggg aaacgcagcg gcgccgaccc tgggtctcgc acattcttca cgtccgttcg
     2641 cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc
     2701 gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc
     2761 cgcacgtctc actagtaccc tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg
     2821 accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga gcagcggccg
     2881 ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc
     2941 gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct ccctcgttga
     3001 ccgaatcacc gacctctctc cccaggggga tccaccggag gccaccatgg tgagcaaggg
     3061 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     3121 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     3181 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     3241 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     3301 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     3361 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     3421 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     3481 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     3541 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     3601 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     3661 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     3721 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggac tcagatctcg
     3781 agctcaagct tcgaattctg cagtcgacgg taccggagtg caggtggaaa ccatctcccc
     3841 aggagacggg cgcaccttcc ccaagcgcgg ccagacctgc gtggtgcact acaccgggat
     3901 gcttgaagat ggaaagaaag ttgattcctc ccgggacaga aacaagccct ttaagtttat
     3961 gctaggcaag caggaggtga tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg
     4021 tcagagagcc aaactgacta tatctccaga ttatgcctat ggtgccactg ggcacccagg
     4081 catcatccca ccacatgcca ctctcgtctt cgatgtggag cttctaaaac tggaagatcc
     4141 aaaaaagaag agaaaggtag aggactaatc tagataactg atcataatca gccataccac
     4201 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     4261 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     4321 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4381 tttgtccaaa ctcatcaatg tatcttaacg cgtcgatcat attcaataac ccttaatggg
     4441 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg
     4501 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
     4561 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
     4621 ttcgcaacgg gtttgccgcc agaacacagg accggttcta gagcgctgcc accatggcca
     4681 agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca
     4741 tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct
     4801 tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg
     4861 gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga
     4921 acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg
     4981 ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat
     5041 tgctgccctc tggttatgtg tgggagggct aagcgggact ctggggttcg aaatgaccga
     5101 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     5161 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     5221 catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat
     5281 accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg
     5341 gtcgttacta gggacaggat tggtgacaga aaagccccat ccttaggcct cctccttcct
     5401 agtctcctga tattgggtct aacccccacc tcctgttagg cagattcctt atctggtgac
     5461 acacccccat ttcctggagc catctctctc cttgccagaa cctctaaggt ttgcttacga
     5521 tggagccaga gaggatcctg ggaggggaat tcactggccg tcgttttaca acgtcgtgac
     5581 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     5641 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     5701 ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5761 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5821 ccaccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5881 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccg
//
