LOCUS       sequence-59186-e        7152 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 7152)
  AUTHORS   Sakuma T, Hosoi S, Woltjen K, Suzuki KI, Kashiwagi K, Wada H, Ochiai
            H, Miyamoto T, Kawai N, Sasakura Y, Matsuura S, Okada Y, Kawahara A,
            Hayashi S, Yamamoto T
  TITLE     Efficient TALEN construction and evaluation methods for human cell 
            and animal applications.
  JOURNAL   Genes Cells. 2013 Feb 6. doi: 10.1111/gtc.12037.
  PUBMED    23388034
REFERENCE   2  (bases 1 to 7152)
  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: "Genes 
            Cells. 2013 Feb 6. doi: 10.1111/gtc.12037."
COMMENT     Sequence Label: pcDNA-TAL-NC2
FEATURES             Location/Qualifiers
     source          1..7152
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..594
                     /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(617..634)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    623..847
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      893..1321
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(980..999)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1190..1211
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1330..1350)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1335..1665
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1516..1651
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1578..1597
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1732..2526
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1786..1805)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2396..2415
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2702..2823
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2739..2758)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2793..2812
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2872..2888)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2896..2912
                     /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)."
     promoter        complement(join(2920..2926,2927..2944,2945..2950))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2965..2986
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3103..3120)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3274..3862)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3354..3373)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4033..4824,4825..4893))
                     /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     4656..4675
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4894..4998)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5073..5092)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5264..5643
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5644..5847
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5798..5818
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        5892..5910
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           5938..5980
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             6060..6080
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6090..6113
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6114..6140
                     /codon_start=1
                     /product="epitope tag from the baculovirus Autographa 
                     californica GP64 envelope fusion protein"
                     /label=AcV5 tag
                     /translation="SWKDASGWS"
     CDS             complement(6504..6911)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TRQCIARCMHCLHCGHAATFCNLHKLACVQL"
     primer_bind     6761..6783
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6775..6792
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6776..6792
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6802..6820
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6868..6888)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6868..6886)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6907..6923)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6931..6947
                     /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)."
     promoter        complement(join(6955..6961,6962..6979,6980..6985))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7000..7021
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 agatcccagc tagtcaaaag tgaactggag gagaagaaat ctgaacttcg tcataaattg
       61 aaatatgtgc ctcatgaata tattgaatta attgaaattg ccagaaattc cactcaggat
      121 agaattcttg aaatgaaggt aatggaattt tttatgaaag tttatggata tagaggtaaa
      181 catttgggtg gatcaaggaa accggacgga gcaatttata ctgtcggatc tcctattgat
      241 tacggtgtga tcgtggatac taaagcttat agcggaggtt ataatctgcc aattggccaa
      301 gcagatgaaa tgcaacgata tgtcgaagaa aatcaaacac gaaacaaaca tatcaaccct
      361 aatgaatggt ggaaagtcta tccatcttct gtaacggaat ttaagttttt atttgtgagt
      421 ggtcacttta aaggaaacta caaagctcag cttacacgat taaatcatat cactaattgt
      481 aatggagctg ttcttagtgt agaagagctt ttaattggtg gagaaatgat taaagccggc
      541 acattaacct tagaggaagt gagaaggaaa tttaataacg gcgagataaa cttttaatag
      601 gaaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
      661 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
      721 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
      781 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
      841 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
      901 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
      961 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1021 ggctttcccc gtcaagctct aaatcggggc atccctttag ggttccgatt tagtgcttta
     1081 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     1141 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     1201 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     1261 ttggggattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     1321 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccaggcaggc
     1381 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     1441 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     1501 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     1561 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     1621 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     1681 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     1741 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     1801 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     1861 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     1921 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     1981 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     2041 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     2101 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     2161 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     2221 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     2281 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     2341 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     2401 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     2461 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     2521 ttctgagcgg gactctgggg ttcgcgaaat gaccgaccaa gcgacgccca acctgccatc
     2581 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     2641 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     2701 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2761 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2821 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     2881 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     2941 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3001 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3061 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3121 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3181 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3241 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3301 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3361 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3421 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg
     3481 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3541 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3601 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3661 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     3721 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3781 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3841 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3901 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3961 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4021 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4081 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4141 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4201 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4261 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4321 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4381 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4441 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4501 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4561 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4621 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4681 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4741 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4801 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4861 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4921 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4981 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     5041 agatctcccg atcccctatg gtcgactctc agtacaatct gctctgatgc cgcatagtta
     5101 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     5161 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     5221 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
     5281 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     5341 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     5401 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     5461 tgggtggact atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     5521 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     5581 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     5641 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
     5701 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
     5761 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
     5821 cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg
     5881 cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagtt aagcttgctt
     5941 gttctttttg cagaagctca gaataaacgc tcaactttgg cagataagct cggatctaac
     6001 tcgagaaaga tattgtatat atcgtaacaa taggaggttc aacaatggct tcctcccctc
     6061 caaagaaaaa gagaaaggtt gcggccgctg actacaagga tgacgacgat aaaagttgga
     6121 aggacgcaag tggttggtct agaatgcatg cggccccgcg acggcgtgct gcgcaaccct
     6181 ccgacgcttc gccggccgcg caggtggatc tacgcacgct cggctacagt cagcagcagc
     6241 aagagaagat caaaccgaag gtgcgttcga cagtggcgca gcaccacgag gcactggtgg
     6301 gccatgggtt tacacacgcg cacatcgttg cgctcagcca acacccggca gcgttaggga
     6361 ccgtcgctgt cacgtatcag cacataatca cggcgttgcc agaggcgaca cacgaagaca
     6421 tcgttggcgt cggcaaacag tggtccggcg cacgcgccct ggaggccttg ctcacggatg
     6481 cgggggagtt gagaggtccg ccgttacagt tggacacagg ccaacttgtg aagattgcaa
     6541 aacgtggcgg cgtgaccgca atggaggcag tgcatgcatc gcgcaatgca ctgacgggtg
     6601 cccccctgga gacgggcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac
     6661 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     6721 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     6781 acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc
     6841 cccctcgagg tcctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     6901 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     6961 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     7021 aattgcgttg cgctcactgc ccgctttcca ccggtcgtct ccaacgacca cctcgtcgcc
     7081 ttggcctgcc tcggcggacg tcctgccatg gatgcagtga aaaagggatt gccgcacgcg
     7141 ccggaattga tc
//
