LOCUS       sequence-178189-        5774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for zinc finger nuclease targeting 
            human AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5774)
  AUTHORS   Zotova A, Lopatukhina E, Filatov A, Khaitov M, Mazurov D
  TITLE     Gene Editing in Human Lymphoid Cells: Role for Donor DNA, Type of 
            Genomic Nuclease and Cell Selection Method.
  JOURNAL   Viruses. 2017 Nov 2;9(11). pii: v9110325. doi: 10.3390/v9110325.
  PUBMED    29099045
REFERENCE   2  (bases 1 to 5774)
  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: "Viruses."; 
            date: "2017-11-2"; volume: "9(11). pii: v9110325. doi"; pages: " 
            10.3390/v9110325"
COMMENT     Sequence Label: pCMV-ZFN-AAVS1
FEATURES             Location/Qualifiers
     source          1..5774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    104..125
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(140..145,146..163,164..170)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    178..194
                     /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     183..205
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     202..218
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     202..218
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        313..692
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        693..896
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     847..867
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     893..917
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1104..1127
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1134..1154
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2133..2186
                     /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"
     CDS             2196..2222
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2229..2249
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3236..3370
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3273..3292)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3327..3346
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3376..3393)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3376..3392)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3385..3407)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3601..3620)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3720..3742
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3780..3798)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3866..3970
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3971..4039,4040..4831)
                     /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(4189..4208)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5002..5590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5491..5510
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5744..5761
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
       61 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
      121 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
      181 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcttggcc
      241 cattgcatac gttgtatcca tatcataata tgtacattta tattggctca tgtccaacat
      301 taccgccatg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      361 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      421 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      481 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      541 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      601 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      661 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      721 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      781 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      841 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt
      901 tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac
      961 accgggaccg atccagcctc cgcggccggg aacggtgcat tggaacgcgg attccccgtg
     1021 ccaagagtga cgtaagtacc gcctatagag tctataggcc cacccccttg gcttcttatg
     1081 cgacggatct gcctgcagcc atggattaca aggatgacga tgacaagggc gcccccaaga
     1141 agaagaggaa ggtgggcatt cacgggaatt ccgccgctat ggctgagagg cccttccagt
     1201 gtcgaatctg catgcgtaac ttcagttaca actggcacct ccagcgccac atccgcaccc
     1261 acacaggcga gaagcctttt gcctgtgaca tttgtgggag gaagtttgcc cgctccgacc
     1321 acctgaccac ccataccaag atacacaccg gttctcagaa gcccttccag tgtcgaatct
     1381 gcatgcgtaa cttcagtcac aactacgccc gcgactgcca catccgcacc cacacaggcg
     1441 agaagccttt tgcctgtgac atttgtggga ggaagtttgc ccagaactcc acccgcatcg
     1501 gccataccaa gatacacctg cggggatccc agctggtgaa gagcgagctg gaggagaaga
     1561 agtccgagct gcggcacaag ctgaagtacg tgccccacga gtacatcgag ctgatcgaga
     1621 tcgccaggaa cagcacccag gaccgcatcc tggagatgaa ggtgatggag ttcttcatga
     1681 aggtgtacgg ctacagggga aagcacctgg gcggaagcag aaagcctgac ggcgccatct
     1741 atacagtggg cagccccatc gattacggcg tgatcgtgga cacaaaggcc tacagcggcg
     1801 gctacaatct gcctatcggc caggccgacg agatggagag atacgtggag gagaaccaga
     1861 cccggaataa gcacctcaac cccaacgagt ggtggaaggt gtaccctagc agcgtgaccg
     1921 agttcaagtt cctgttcgtg agcggccact tcaagggcaa ctacaaggcc cagctgacca
     1981 ggctgaacca catcaccaac tgcaatggcg ccgtgctgag cgtggaggag ctgctgatcg
     2041 gcggcgagat gatcaaagcc ggcaccctga cactggagga ggtgcggcgc aagttcaaca
     2101 acggcgagat caacttcaga tctggcggcg gagagggcag aggaagtctt ctaacatgcg
     2161 gtgacgtgga ggagaatccc ggccctagga tgggctaccc atacgatgtt ccagattacg
     2221 ctggcgcccc caagaagaag aggaaggtgg gcattcacgg tctagacgcc gctatggctg
     2281 agaggccctt ccagtgtcga atctgcatgc gtaacttcag tcagtcctcc aacctggccc
     2341 gccacatccg cacccacaca ggcgagaagc cttttgcctg tgacatttgt gggaggaagt
     2401 ttgcccgcac cgactacctg gtggaccata ccaagataca caccggttct cagaagccct
     2461 tccagtgtcg aatctgcatg cgtaacttca gttacaacac ccacctgacc cgccacatcc
     2521 gcacccacac aggcgagaag ccttttgcct gtgacatttg tgggaggaag tttgcccagg
     2581 gctacaacct ggccggccat accaagatac acctgcgggc tagccagctg gtgaagagcg
     2641 agctggagga gaagaagtcc gagctgcggc acaagctgaa gtacgtgccc cacgagtaca
     2701 tcgagctgat cgagatcgcc aggaacagca cccaggaccg catcctggag atgaaggtga
     2761 tggagttctt catgaaggtg tacggctaca ggggaaagca cctgggcgga agcagaaagc
     2821 ctgacggcgc catctataca gtgggcagcc ccatcgatta cggcgtgatc gtggacacaa
     2881 aggcctacag cggcggctac aatctgccta tcggccaggc cgacgagatg cagagatacg
     2941 tgaaggagaa ccagacccgg aataagcaca tcaaccccaa cgagtggtgg aaggtgtacc
     3001 ctagcagcgt gaccgagttc aagttcctgt tcgtgagcgg ccacttcaag ggcaactaca
     3061 aggcccagct gaccaggctg aaccacaaaa ccaactgcaa tggcgccgtg ctgagcgtgg
     3121 aggagctgct gatcggcggc gagatgatca aagccggcac cctgacactg gaggaggtgc
     3181 ggcgcaagtt caacaacggc gagatcaact tctgataagg taccgagctc gaattaactt
     3241 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3301 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     3361 tgtctggatc aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     3421 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     3481 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     3541 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     3601 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     3661 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3721 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     3781 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     3841 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3901 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3961 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4021 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4081 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4141 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4201 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     4261 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4321 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4381 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4441 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4501 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4561 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4621 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4681 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4741 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4801 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4861 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4921 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4981 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5041 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5101 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     5161 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5221 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5281 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5341 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5401 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5461 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5521 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5581 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5641 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     5701 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     5761 ggaagcggaa gagc
//
