LOCUS       sequence-75858-e        6978 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 6978)
  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 6978)
  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-153/47-VR-NI
FEATURES             Location/Qualifiers
     source          1..6978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(212..232)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        217..546
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      397..532
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     459..478
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             595..1620
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             595..1620
                     /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    1750..1871
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1787..1806)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1841..1860
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(1924..2715,2716..2784))
                     /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     2547..2566
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2785..2876)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2918..2937
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2943..2962
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3048..3636
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3537..3556
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3790..3807
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3906..3922
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4378..4394
                     /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        4421..4800
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4801..5004
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4955..4975
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        5049..5067
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           5075..5117
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      5125..5134
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5146..5166
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5176..5199
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5200..5226
                     /codon_start=1
                     /product="epitope tag from the baculovirus Autographa 
                     californica GP64 envelope fusion protein"
                     /label=AcV5 tag
                     /translation="SWKDASGWS"
     CDS             complement(5590..6000)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLLEG
                     CSRERIARCMHCLHCGHAATFCNLHKLACVQL"
     primer_bind     5850..5872
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5865..5881
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5891..5909
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5957..5977)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5957..5975)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5996..6012)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5996..6012)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6009..6031)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6020..6036
                     /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(6044..6050,6051..6068,6069..6074))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6089..6110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             6356..6943
                     /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(6955..6972)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(6961..6978,1..94)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
       61 ctgtcctttc ctaataaaat gaggaaattg catcacaaca ctcaacccta tctcggtcta
      121 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
      181 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
      241 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
      301 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
      361 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
      421 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
      481 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
      541 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgatgaaa
      601 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtt
      661 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
      721 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
      781 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
      841 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
      901 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
      961 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     1021 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     1081 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     1141 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     1201 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     1261 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     1321 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     1381 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     1441 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     1501 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     1561 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag
     1621 cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     1681 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     1741 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     1801 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     1861 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     1921 tcattaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     1981 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     2041 ccccagcgct gcgatgatac cgcgagaacc acgctcaccg gctccggatt tatcagcaat
     2101 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     2161 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     2221 caacgttgtt gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     2281 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     2341 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     2401 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     2461 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     2521 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     2581 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     2641 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     2701 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     2761 gacacggaaa tgttgaatac tcatattctt cctttttcaa tattattgaa gcatttatca
     2821 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     2881 ggtcagtgtt acaaccaatt aaccaattct gaacattatc gcgagcccat ttatacctga
     2941 atatggctca taacacccct tgctcatgac caaaatccct taacgtgagt tacgcgcgcg
     3001 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3061 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3121 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3181 ataccaaata ctgttcttct agtgtagccg tagttagccc accacttcaa gaactctgta
     3241 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3301 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3361 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3421 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3481 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3541 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3601 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3661 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3721 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3781 accgagcgca gcgagtcagt gagcgaggaa gcggaaggcg agagtaggga actgccaggc
     3841 atcaaactaa gcagaaggcc cctgacggat ggcctttttg cgtttctaca aactctttct
     3901 gtgttgtaaa acgacggcca gtcttaagct cgggccccct gggcggttct gataacgagt
     3961 aatcgttaat ccgcaaataa cgtaaaaacc cgcttcggcg ggttttttta tggggggagt
     4021 ttagggaaag agcatttgtc agaatattta agggcgcctg tcactttgct tgatatatga
     4081 gaattattta accttataaa tgagaaaaaa gcaacgcact ttaaataaga tacgttgctt
     4141 tttcgattga tgaacaccta taattaaact attcatctat tatttatgat tttttgtata
     4201 tacaatattt ctagtttgtt aaagagaatt aagaaaataa atctcgaaaa taataaaggg
     4261 aaaatcagtt tttgatatca aaattataca tgtcaacgat aatacaaaat ataatacaaa
     4321 ctataagatg ttatcagtat ttattatcat ttagaataaa ttttgtgtcg cccttaattg
     4381 tgagcggata acaattacga gcttcatgca cagtggcgtt gacattgatt attgactagt
     4441 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     4501 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     4561 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     4621 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     4681 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     4741 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     4801 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     4861 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     4921 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     4981 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
     5041 atcgaaatta atacgactca ctatagggaa gcttcttgtt ctttttgcag aagctcagaa
     5101 taaacgctca actttggcct cgaggccacc atggcttcct cccctccaaa gaaaaagaga
     5161 aaggttgcgg ccgctgacta caaggatgac gacgataaaa gttggaagga cgcaagtggt
     5221 tggtctagaa tgcatgcggc cccgcgacgg cgtgctgcgc aaccctccga cgcttcgccg
     5281 gccgcgcagg tggatctacg cacgctcggc tacagtcagc agcagcaaga gaagatcaaa
     5341 ccgaaggtgc gttcgacagt ggcgcagcac cacgaggcac tggtgggcca tgggtttaca
     5401 cacgcgcaca tcgttgcgct cagccaacac ccggcagcgt tagggaccgt cgctgtcacg
     5461 tatcagcaca taatcacggc gttgccagag gcgacacacg aagacatcgt tggcgtcggc
     5521 aaacagtggt ccggcgcacg cgccctggag gccttgctca cggatgcggg ggagttgaga
     5581 ggtccgccgt tacagttgga cacaggccaa cttgtgaaga ttgcaaaacg tggcggcgtg
     5641 accgcaatgg aggcagtgca tgcatcgcgc aatgcgctca cgggagcacc cctcaaggag
     5701 acgggcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg
     5761 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     5821 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     5881 tgagcgcgcg taatacgact cactataggg cgaattgggt accgggcccc ccctcgaggt
     5941 cctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     6001 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     6061 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     6121 gctcactgcc cgctttccac cggtcgtctc cacccctgag caggtagtgg ctattgcatc
     6181 caacataggg ggcagacccg cactggagtc aatcgtggcc cagctttcga ggccggaccc
     6241 cgcgctggcc gcactcacta atgatcatct tgtagcgctg gcctgcctcg gcggacgtcc
     6301 tgccatggat gcagtgaaaa agggattgcc gcacgcgccg gaattgatca gatcccagct
     6361 agtgaaatct gaattggaag agaagaaatc tgaacttaga cataaattga aatatgtgcc
     6421 acatgaatat attgaattga ttgaaatcgc aagaaattca actcaggata gaatccttga
     6481 aatgaaggtg atggagttct ttatgaaggt ttatggttat cgtggtaaac atttgggtgg
     6541 atcaaggaaa ccagacggag caatttatac tgtcggatct cctattgatt acggtgtgat
     6601 cgttgatact aaggcatatt caggaggtta taatcttcca attggtcaag cagatgaaat
     6661 gcaaagatat gtcgaagaga atcaaacaag aaacaagcat atcaacccta atgaatggtg
     6721 gaaagtctat ccatcttcag taacagaatt taagttcttg tttgtgagtg gtcatttcaa
     6781 aggaaactac aaagctcagc ttacaagatt gaatcatatc actaattgta atggagctgt
     6841 tcttagtgta gaagagcttt tgattggtgg agaaatgatt aaagctggta cattgacact
     6901 tgaggaagtg agaaggaaat ttaataacgg tgagataaac ttttaaaaaa tcagcctcga
     6961 ctgtgccttc tagttgcc
//
