LOCUS       sequence-97508-e        7354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  two TALEN monomers in one plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7354)
  AUTHORS   Mariano A, Xu L, Han R
  TITLE     Highly efficient genome editing via 2A-coupled co-expression of two 
            TALEN monomers.
  JOURNAL   BMC Res Notes. 2014 Sep 10;7:628. doi: 10.1186/1756-0500-7-628.
  PUBMED    25204665
REFERENCE   2  (bases 1 to 7354)
  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: "BMC Res 
            Notes."; date: "2014-09-10"; volume: "7:628. doi"; pages: " 
            10.1186/1756-0500-7-628"
COMMENT     Sequence Label: PTAL10
FEATURES             Location/Qualifiers
     source          1..7354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /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(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1330..1389
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     CDS             1399..1464
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1471..1491
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2080..2667
                     /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="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKV
                     MEFFMKVYGYRGEHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMER
                     YVEENQTRDKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             2683..2736
                     /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             2743..2769
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2773..2799
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2806..2826
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3416..4003
                     /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="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKV
                     MEFFMKVYGYRGEHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVKENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNRKTNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     polyA_signal    4142..4263
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4179..4198)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4233..4252
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4270..4725)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4407..4428)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4619..4638
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4752..4856
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4858..5215
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4858..5049
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4881..4901)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5066..5201
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5250..6044
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5250..6044
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5304..5323)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5914..5933
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6232..6251)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6276..6323
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6652..7240
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7141..7160
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagc aatgtactaa ctacgctttg ttgaaactcg ctggcgatgt tgaaagtaac
     1381 cccggtcctg aattcatgga ctacaaagac catgacggtg attataaaga tcatgacatc
     1441 gattacaagg atgacgatga caagatggcc cccaagaaga agaggaaggt gggcattcac
     1501 ggggtaccta tggtggatct acgcacgctc ggctacagtc agcagcagca agagaagatc
     1561 aaaccgaagg tgcgttcgac agtggcgcag caccacgagg cactggtggg ccatgggttt
     1621 acacacgcgc acatcgttgc gctcagccaa cacccggcag cgttagggac cgtcgctgtc
     1681 acgtatcagc acataatcac ggcgttgcca gaggcgacac acgaagacat cgttggcgtc
     1741 ggcaaacagt ggtccggcgc acgcgccctg gaggccttgc tcacggatgc gggggagttg
     1801 agaggtccgc cgttacagtt ggacacaggc caacttgtga agattgcaaa acgtggcggc
     1861 gtgaccgcaa tggaggcagt gcatgcatcg cgcaatgcac tgacgggtgc tcccctggag
     1921 acgtcgaccg tctccaacga ccacctcgtc gccttggcct gcctcggcgg acgtcctgcc
     1981 atggatgcag tgaaaaaggg attgccgcac gcgccggaat tgatcagaag agtcaatcgc
     2041 cgtattggcg aacgcacgtc ccatcgcgtt gccggatccc agctggtcaa aagtgaactg
     2101 gaggagaaga aatctgaact tcgtcataaa ttgaaatatg tgcctcatga atatattgaa
     2161 ttaattgaaa ttgccagaaa tcccactcag gataggattc ttgaaatgaa ggtaatggaa
     2221 ttttttatga aagtttatgg atatagaggt gaacatttgg gtggatcaag gaaaccggac
     2281 ggagcaattt atactgtcgg atctcctatt gattacggtg tgatcgtgga tactaaagct
     2341 tatagcggag gttataatct gccaattggc caagcagatg aaatggaacg atatgtcgaa
     2401 gaaaatcaaa cacgagacaa acatctcaac cctaatgaat ggtggaaagt ctatccatct
     2461 tctgtaacgg aatttaagtt tttatttgtg agtggtcact ttaaaggaaa ctacaaagct
     2521 cagcttacac gattaaatca tatcactaat tgtaatggag ctgttcttag tgtagaagag
     2581 cttttaattg gtggagaaat gattaaagcc ggcacattaa ccttagagga agtgagaagg
     2641 aaatttaata acggcgagat aaactttaga tctggcggcg gagagggcag aggaagtctt
     2701 ctaacatgcg gtgacgtgga ggagaatccc ggccctagga tgtacccata cgatgttcca
     2761 gattacgcta tgtacccata cgatgttcca gattacgcta tggcccccaa gaagaagagg
     2821 aaggtgggca ttcacggggt acctatggtg gatctacgca cgctcggcta cagtcagcag
     2881 cagcaagaga agatcaaacc gaaggtgcgt tcgacagtgg cgcagcacca cgaggcactg
     2941 gtgggccatg ggtttacaca cgcgcacatc gttgcgctca gccaacaccc ggcagcgtta
     3001 gggaccgtcg ctgtcacgta tcagcacata atcacggcgt tgccagaggc gacacacgaa
     3061 gacatcgttg gcgtcggcaa acagtggtcc ggcgcacgcg ccctggaggc cttgctcacg
     3121 gatgcggggg agttgagagg tccgccgtta cagttggaca caggccaact tgtgaagatt
     3181 gcaaaacgtg gcggcgtgac cgcaatggag gcagtgcatg catcgcgcaa tgcactgacg
     3241 ggtgctcccc tggagaccgt cgacggtctc caacgaccac ctcgtcgcct tggcctgcct
     3301 cggcggacgt cctgccatgg atgcagtgaa aaagggattg ccgcacgcgc cggaattgat
     3361 cagaagagtc aatcgccgta ttggcgaacg cacgtcccat cgcgttgccg gatcccagct
     3421 ggtcaaaagt gaactggagg agaagaaatc tgaacttcgt cataaattga aatatgtgcc
     3481 tcatgaatat attgaattaa ttgaaattgc cagaaatccc actcaggata ggattcttga
     3541 aatgaaggta atggaatttt ttatgaaagt ttatggatat agaggtgaac atttgggtgg
     3601 atcaaggaaa ccggacggag caatttatac tgtcggatct cctattgatt acggtgtgat
     3661 cgtggatact aaagcttata gcggaggtta taatctgcca attggccaag cagatgaaat
     3721 gcaacgatat gtcaaggaaa atcaaacacg aaacaaacat atcaacccta atgaatggtg
     3781 gaaagtctat ccatcttctg taacggaatt taagttttta tttgtgagtg gtcactttaa
     3841 aggaaactac aaagctcagc ttacacgatt aaatcgtaag actaattgta atggagctgt
     3901 tcttagtgta gaagagcttt taattggtgg agaaatgatt aaagccggca cattaacctt
     3961 agaggaagtg agaaggaaat ttaataacgg cgagataaac ttttaactcg agtgatccac
     4021 cggatctaga taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt
     4081 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     4141 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4201 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4261 ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     4321 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     4381 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     4441 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     4501 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     4561 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     4621 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     4681 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     4741 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4801 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     4861 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     4921 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     4981 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     5041 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     5101 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     5161 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     5221 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     5281 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     5341 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     5401 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     5461 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     5521 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     5581 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     5641 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     5701 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     5761 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     5821 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     5881 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     5941 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     6001 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     6061 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     6121 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     6181 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     6241 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     6301 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     6361 cgatacccca ccgagacacc attggggcca atacgcccgc gtttcttcct tttccccacc
     6421 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     6481 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     6541 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6601 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6661 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6721 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6781 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6841 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6901 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6961 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7021 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7081 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7141 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7201 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7261 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7321 tgattctgtg gataaccgta ttaccgccat gcat
//
