LOCUS       sequence-305024-        5413 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutisite Gateway mediated vector construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5413)
  AUTHORS   Yoshimatsu S, Sone T, Nakajima M, Sato T, Okochi R, Ishikawa M, 
            Nakamura M, Sasaki E, Shiozawa S, Okano H
  TITLE     A versatile toolbox for knock-in gene targeting based on the 
            Multisite Gateway technology.
  JOURNAL   PLoS One. 2019 Aug 27;14(8):e0221164. doi: 
            10.1371/journal.pone.0221164. eCollection 2019.
  PUBMED    31454364
REFERENCE   2  (bases 1 to 5413)
  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: "PLoS One.";
            date: "2019-08-27"; volume: "14(8):e0221164. doi"; pages: " 
            10.1371/journal.pone.0221164. eCollection 2019"
COMMENT     Sequence Label: pATD
FEATURES             Location/Qualifiers
     source          1..5413
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(55..123,124..915)
                     /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(273..292)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1086..1674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1575..1594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1779..1902
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1927..1932,1933..1950,1951..1957)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2011..2691
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(2077..2096)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3011..3316
                     /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     3195..3214
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3357..3480)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3515..4014
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3550..3573)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3947..3966
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     5'UTR           4056..4095
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     CDS             4113..5243
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL
                     LQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     primer_bind     complement(5241..5260)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5285..5333
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        join(5363..5413,1..54)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
       61 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      121 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      181 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      241 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      301 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
      361 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
      421 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
      481 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
      541 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
      601 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
      661 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
      721 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
      781 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
      841 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
      901 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
      961 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1021 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1081 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1141 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1201 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     1261 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1321 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     1381 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     1441 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     1501 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     1561 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     1621 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     1681 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtgcggc
     1741 cgcgccggca tgcctgcagg tcgggatatc tgaattatca actttgtata ataaagttga
     1801 acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca
     1861 gactacataa tactgtaaaa cacaacatat ccagtcacta tggcggccgc attaggcacc
     1921 ccaggcttta cactttatgc ttccggctcg tataatgtgt ggattttgag ttaggatccg
     1981 tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc
     2041 gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa
     2101 tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa
     2161 aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat
     2221 ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct
     2281 tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac
     2341 gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac
     2401 ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg
     2461 gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt
     2521 ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag
     2581 gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag
     2641 tactgcgatg agtggcaggc ggggcgtaat ctagaggatc cggcttacta aaagccagat
     2701 aacagtatgc gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata
     2761 cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt attacagtga cagttgacag
     2821 cgacagctat cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca
     2881 accatgcaga atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa
     2941 gggatggctg aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg
     3001 ggctggtgaa atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt
     3061 tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc
     3121 cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg
     3181 ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg
     3241 ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat
     3301 gttctgggga atataaatgt caggctccct tatacacagc cagtctgcag gtcgaccata
     3361 gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat
     3421 ttaatatatt gatatttata tcattttacg tttctcgttc aacttttcta tacaaagttg
     3481 atagcttgat atcaatactc tagagaaact cgaggggtag gggaggcgct tttcccaagg
     3541 cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc
     3601 tggcctcgca cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc
     3661 cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc
     3721 gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac
     3781 agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc
     3841 tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc
     3901 gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt
     3961 caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgacctgc
     4021 agcgacccgc ttaacagcgt caacagcgtg ccgcagatct tggtggcgtg aaactcccgc
     4081 acctcttcgg caagcgcctt gtagaagcgc gtatggcttc gtacccctgc catcaacacg
     4141 cgtctgcgtt cgaccaggct gcgcgttctc gcggccatag caaccgacgt acggcgttgc
     4201 gccctcgccg gcagcaagaa gccacggaag tccgcctgga gcagaaaatg cccacgctac
     4261 tgcgggttta tatagacggt cctcacggga tggggaaaac caccaccacg caactgctgg
     4321 tggccctggg ttcgcgcgac gatatcgtct acgtacccga gccgatgact tactggcagg
     4381 tgctgggggc ttccgagaca atcgcgaaca tctacaccac acaacaccgc ctcgaccagg
     4441 gtgagatatc ggccggggac gcggcggtgg taatgacaag cgcccagata acaatgggca
     4501 tgccttatgc cgtgaccgac gccgttctgg ctcctcatat cgggggggag gctgggagct
     4561 cacatgcccc gcccccggcc ctcaccctca tcttcgaccg ccatcccatc gccgccctcc
     4621 tgtgctaccc ggccgcgcga taccttatgg gcagcatgac cccccaggcc gtgctggcgt
     4681 tcgtggccct catcccgccg accttgcccg gcacaaacat cgtgttgggg gcccttccgg
     4741 aggacagaca catcgaccgc ctggccaaac gccagcgccc cggcgagcgg cttgacctgg
     4801 ctatgctggc cgcgattcgc cgcgtttacg ggctgcttgc caatacggtg cggtatctgc
     4861 agggcggcgg gtcgtggcgg gaggattggg gacagctttc ggggacggcc gtgccgcccc
     4921 agggtgccga gccccagagc aacgcgggcc cacgacccca tatcggggac acgttattta
     4981 ccctgtttcg ggcccccgag ttgctggccc ccaacggcga cctgtacaac gtgtttgcct
     5041 gggccttgga cgtcttggcc aaacgcctcc gtcccatgca cgtctttatc ctggattacg
     5101 accaatcgcc cgccggctgc cgggacgccc tgctgcaact tacctccggg atggtccaga
     5161 cccacgtcac cacccccggc tccataccga cgatctgcga cctggcgcgc acgtttgccc
     5221 gggagatggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     5281 atgacggcaa taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg ttccctcagg
     5341 tttcctaggc acgtagtgtt ttcgcggaac ccctatttgt ttatttttct aaatacattc
     5401 aaatatgtat ccg
//
