LOCUS       sequence-58121-e        6542 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6542)
  AUTHORS   Cong L, Zhou R, Kuo YC, Cunniff M, Zhang F
  TITLE     Comprehensive interrogation of natural TALE DNA-binding modules and 
            transcriptional repressor domains.
  JOURNAL   Nat Commun. 2012 Jul 24;3:968. doi: 10.1038/ncomms1962.
  PUBMED    22828628
REFERENCE   2  (bases 1 to 6542)
  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: "2012-07-24"; volume: "3:968. doi"; pages: " 
            10.1038/ncomms1962"
COMMENT     Sequence Label: pAAV_TALEN-FokI(WT)-BB_V3
FEATURES             Location/Qualifiers
     source          1..6542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     enhancer        160..463
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        465..742
                     /label=chicken beta-actin promoter
     intron          743..971
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      989..998
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             998..1063
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1073..1093
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        join(1538..1542,1543..1560,1561..1567)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1621..2280
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1687..1706)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2616..2921
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2800..2819
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2962..3549
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     primer_bind     complement(3583..3600)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3589..3796
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3805..3945
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3805..3934
                     /label=AAV2 ITR
     rep_origin      4020..4475
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4107..4126)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4317..4338
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4492..4511)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4611..4633
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4671..4689)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4757..4861
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4862..4930,4931..5722)
                     /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(5080..5099)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5893..6481
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6382..6401
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggcctct agaggtaccc gttacataac ttacggtaaa
      181 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      241 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      301 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      361 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      421 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
      481 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
      541 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg
      601 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca
      661 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
      721 aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc gtgccccgct
      781 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
      841 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
      901 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
      961 ctttttttca ggttggaccg gtggatccgc caccatggac tataaggacc acgacggaga
     1021 ctacaaggat catgatattg attacaaaga cgatgacgat aagaagcttg ccccaaagaa
     1081 gaagcggaag gtcggtatcc acggagtccc agcagccgta gatttgagaa ctttgggata
     1141 ttcacagcag cagcaggaaa agatcaagcc caaagtgagg tcgacagtcg cgcagcatca
     1201 cgaagcgctg gtgggtcatg ggtttacaca tgcccacatc gtagccttgt cgcagcaccc
     1261 tgcagccctt ggcacggtcg ccgtcaagta ccaggacatg attgcggcgt tgccggaagc
     1321 cacacatgag gcgatcgtcg gtgtggggaa acagtggagc ggagcccgag cgcttgaggc
     1381 cctgttgacg gtcgcgggag agctgagagg gcctcccctt cagctggaca cgggccagtt
     1441 gctgaagatc gcgaagcggg gaggagtcac ggcggtcgag gcggtgcacg cgtggcgcaa
     1501 tgcgctcacg ggagcacccc tcaacctgac agagacctta cactttatgc ttccggctcg
     1561 tataatgtgt ggattttgag ttaggttccg tcgagatttt caggagctaa ggaagctaaa
     1621 atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa
     1681 cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat
     1741 attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt
     1801 cacattcttg cccgcctgat gaatgctcat ccggagttcc gtatggcaat gaaagacggt
     1861 gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa
     1921 acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat
     1981 tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag
     2041 aatatgtttt tcgtatcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg
     2101 gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc
     2161 gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat
     2221 gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa
     2281 agatctggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt
     2341 ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc
     2401 agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt
     2461 caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga
     2521 acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa
     2581 cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata
     2641 aaagagagag ccgttatcgt ctgtttgtgg acgtacagag tgatattatt gacacgcccg
     2701 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg
     2761 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     2821 ccagtgtgcc ggtatccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     2881 acatcaaaaa cgccattaac ctgatgttct ggggaatata aggtctctcc cgagaggact
     2941 agtcatcgag tggcaggttc ccaactcgtg aagagtgaac ttgaggagaa aaagtcggag
     3001 ctgcggcaca aattgaaata cgtaccgcat gaatacatcg aacttatcga aattgctagg
     3061 aactcgactc aagacagaat ccttgagatg aaggtaatgg agttctttat gaaggtttat
     3121 ggataccgag ggaagcatct cggtggatca cgaaaacccg acggagcaat ctatacggtg
     3181 gggagcccga ttgattacgg agtgatcgtc gacacgaaag cctacagcgg tgggtacaat
     3241 cttcccatcg ggcaggcaga tgagatgcaa cgttatgtcg aagaaaatca gaccaggaac
     3301 aaacacatca atccaaatga gtggtggaaa gtgtatcctt catcagtgac cgagtttaag
     3361 tttttgtttg tctctgggca tttcaaaggc aactataagg cccagctcac acggttgaat
     3421 cacattacga actgcaatgg tgcggttttg tccgtagagg aactgctcat tggtggagaa
     3481 atgatcaaag cgggaactct gacactggaa gaagtcagac gcaagtttaa caatggcgag
     3541 atcaatttcc gctcataaga attcctagag ctcgctgatc agcctcgact gtgccttcta
     3601 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3661 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3721 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata
     3781 gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     3841 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     3901 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     3961 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     4021 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4081 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4141 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4201 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     4261 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4321 actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata
     4381 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4441 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     4501 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     4561 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4621 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     4681 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4741 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4801 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4861 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4921 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4981 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5041 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5101 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5161 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5221 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5281 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5341 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5401 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5461 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5521 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag
     5581 ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5641 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5701 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5761 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5821 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5881 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5941 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6001 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     6061 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6121 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6181 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6241 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6301 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6361 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6421 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6481 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6541 gt
//
