LOCUS       sequence-75854-e        6993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Final destination vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6993)
  AUTHORS   Sakuma T, Ochiai H, Kaneko T, Mashimo T, Tokumasu D, Sakane Y, 
            Suzuki K, Miyamoto T, Sakamoto N, Matsuura S, Yamamoto T
  TITLE     Repeating pattern of non-RVD variations in DNA-binding modules 
            enhances TALEN activity.
  JOURNAL   Sci Rep. 2013 Nov 29;3:3379. doi: 10.1038/srep03379.
  PUBMED    24287550
REFERENCE   2  (bases 1 to 6993)
  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: "Sci Rep."; 
            date: "2013-11-29"; volume: "3:3379. doi"; pages: " 
            10.1038/srep03379"
COMMENT     Sequence Label: ptCMV-136/63-VR-NI
FEATURES             Location/Qualifiers
     source          1..6993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             247..834
                     /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(852..869)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    858..969
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1087..1107)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1092..1421
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1272..1407
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1334..1353
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1470..2495
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1470..2495
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    2625..2746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2662..2681)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2716..2735
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(2799..3590,3591..3659))
                     /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     3422..3441
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3660..3751)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3793..3812
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3818..3837
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3923..4511
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4412..4431
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4665..4682
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4781..4797
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5253..5269
                     /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)."
     enhancer        5296..5675
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5676..5879
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5830..5850
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        5924..5942
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           5950..5992
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      6000..6009
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6009..6074
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6081..6101
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(6150..6836)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLLEG
                     CSRERIVPRVHRLDRRDSSPLRDLQQLARVQLKGRPSQLSRDRQQGLKRSGSAPLFPHT
                     DDRLMCGFRQRRNHVLVLDGDRAKGCRVLRQGYDVGMCKPMTHQRFVMLRDCRPHFGLD
                     LFLLLL"
     primer_bind     6686..6708
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6701..6717
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6727..6745
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6793..6813)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6793..6811)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6832..6848)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6832..6848)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6845..6867)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6856..6872
                     /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(6880..6886,6887..6904,6905..6910))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6925..6946
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gagcaggtag tggctattgc atccaacata gggggcagac ccgcactgga gtcaatcgtg
       61 gcccagcttt cgaggccgga ccccgcgctg gccgcactca ctaatgatca tcttgtagcg
      121 ctggcctgcc tcggcggacg acccgccttg gatgcggtga agaaggggct cccgcacgcg
      181 cctgcattga ttaagcggac caacagaagg atccccgaga ggacatcaca tcgagtggca
      241 ggttcccaac tcgtgaagag tgaacttgag gagaaaaagt cggagctgcg gcacaaattg
      301 aaatacgtac cgcatgaata catcgaactt atcgaaattg ctaggaactc gactcaagac
      361 agaatccttg agatgaaggt aatggagttc tttatgaagg tttatggata ccgagggaag
      421 catctcggtg gatcacgaaa acccgacgga gcaatctata cggtggggag cccgattgat
      481 tacggagtga tcgtcgacac gaaagcctac agcggtgggt acaatcttcc catcgggcag
      541 gcagatgaga tgcaacgtta tgtcgaagaa aatcagacca ggaacaaaca catcaatcca
      601 aatgagtggt ggaaagtgta tccttcatca gtgaccgagt ttaagttttt gtttgtctct
      661 gggcatttca aaggcaacta taaggcccag ctcacacggt tgaatcacat tacgaactgc
      721 aatggtgcgg ttttgtccgt agaggaactg ctcattggtg gagaaatgat caaagcggga
      781 actctgacac tggaagaagt cagacgcaag tttaacaatg gcgagatcaa tttccgctca
      841 taaaaaatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
      901 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
      961 aattgcatca caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     1021 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     1081 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     1141 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     1201 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     1261 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     1321 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     1381 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     1441 tatccatttt cggatctgat cagcacgtga tgaaaaagcc tgaactcacc gcgacgtctg
     1501 tcgagaagtt tctgatcgaa aagttcgaca gcgtttccga cctgatgcag ctctcggagg
     1561 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     1621 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     1681 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
     1741 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     1801 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     1861 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     1921 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     1981 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     2041 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     2101 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     2161 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     2221 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     2281 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     2341 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     2401 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     2461 gacgccccag cactcgtccg agggcaaagg aatagcacgt gctacgagat ttcgattcca
     2521 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     2581 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     2641 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2701 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     2761 cgtcgacctc tagctagagc ttggcgtaat catggtcatt accaatgctt aatcagtgag
     2821 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     2881 tagataacta cgatacggga gggcttacca tctggcccca gcgctgcgat gataccgcga
     2941 gaaccacgct caccggctcc ggatttatca gcaataaacc agccagccgg aagggccgag
     3001 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3061 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat cgctacaggc
     3121 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3181 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3241 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3301 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3361 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3421 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3481 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3541 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3601 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3661 ttcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3721 atatttgaat gtatttagaa aaataaacaa ataggggtca gtgttacaac caattaacca
     3781 attctgaaca ttatcgcgag cccatttata cctgaatatg gctcataaca ccccttgctc
     3841 atgaccaaaa tcccttaacg tgagttacgc gcgcgtcgtt ccactgagcg tcagaccccg
     3901 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3961 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4021 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4081 agccgtagtt agcccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4141 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4201 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4261 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4321 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4381 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4441 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4501 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4561 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4621 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4681 aggaagcgga aggcgagagt agggaactgc caggcatcaa actaagcaga aggcccctga
     4741 cggatggcct ttttgcgttt ctacaaactc tttctgtgtt gtaaaacgac ggccagtctt
     4801 aagctcgggc cccctgggcg gttctgataa cgagtaatcg ttaatccgca aataacgtaa
     4861 aaacccgctt cggcgggttt ttttatgggg ggagtttagg gaaagagcat ttgtcagaat
     4921 atttaagggc gcctgtcact ttgcttgata tatgagaatt atttaacctt ataaatgaga
     4981 aaaaagcaac gcactttaaa taagatacgt tgctttttcg attgatgaac acctataatt
     5041 aaactattca tctattattt atgatttttt gtatatacaa tatttctagt ttgttaaaga
     5101 gaattaagaa aataaatctc gaaaataata aagggaaaat cagtttttga tatcaaaatt
     5161 atacatgtca acgataatac aaaatataat acaaactata agatgttatc agtatttatt
     5221 atcatttaga ataaattttg tgtcgccctt aattgtgagc ggataacaat tacgagcttc
     5281 atgcacagtg gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     5341 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     5401 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     5461 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     5521 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     5581 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     5641 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     5701 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     5761 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     5821 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     5881 tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg actcactata
     5941 gggaagcttc ttgttctttt tgcagaagct cagaataaac gctcaacttt ggcctcgagg
     6001 ccaccatgga ctataaggac cacgacggag actacaagga tcatgatatt gattacaaag
     6061 acgatgacga taagatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag
     6121 cagccgtaga tttgagaact ttgggatatt cacagcagca gcaggaaaag atcaagccca
     6181 aagtgaggtc gacagtcgcg cagcatcacg aagcgctggt gggtcatggg tttacacatg
     6241 cccacatcgt agccttgtcg cagcaccctg cagcccttgg cacggtcgcc gtcaagtacc
     6301 aggacatgat tgcggcgttg ccggaagcca cacatgaggc gatcgtcggt gtggggaaac
     6361 agtggagcgg agcccgagcg cttgaggccc tgttgacggt cgcgggagag ctgagagggc
     6421 ctccccttca gctggacacg ggccagttgc tgaagatcgc gaagcgggga ggagtcacgg
     6481 cggtcgaggc ggtgcacgcg tggcacaatg cgctcacggg agcacccctc aaggagacgg
     6541 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     6601 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     6661 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag
     6721 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcctc
     6781 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
     6841 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     6901 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     6961 actgcccgct ttccaccggt cgtctccacc cct
//
