LOCUS       sequence-304980-        5973 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 5973)
  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 5973)
  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: pUC-DTA-DEST-R3R4(R)
FEATURES             Location/Qualifiers
     source          1..5973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        14..513
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(49..72)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     446..465
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             540..1112
                     /codon_start=1
                     /gene="Corynebacterium diphtheriae tox"
                     /product="diphtheria toxin fragment A"
                     /label=DTA
                     /translation="DDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYD
                     DDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKEL
                     GLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEI
                     NFETRGKRGQDAMYEYMAQACAGNRVRR"
     intron          1189..1254
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     primer_bind     complement(1322..1339)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1328..1552
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1622..1638)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1622..1638)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1635..1657)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1646..1662
                     /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(1670..1676,1677..1694,1695..1700))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1715..1736
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1853..1870)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2024..2612)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2104..2123)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2783..3574,3575..3643))
                     /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     3406..3425
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3644..3748)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3816..3834
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3872..3894)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3994..4013
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4207..4229
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4221..4238
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4222..4238
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4262..4385
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(4410..4415,4416..4433,4434..4440)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             4494..5174
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(4560..4579)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             5494..5799
                     /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     5678..5697
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5840..5963)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 atcgaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
       61 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
      121 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
      181 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
      241 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
      301 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
      361 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
      421 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
      481 ctgttctcct cttcctcatc tccgggcctt tcgacctgca ggtcctcgcc atggatcctg
      541 atgatgttgt tgattcttct aaatcttttg tgatggaaaa cttttcttcg taccacggga
      601 ctaaacctgg ttatgtagat tccattcaaa aaggtataca aaagccaaaa tctggtacac
      661 aaggaaatta tgacgatgat tggaaagggt tttatagtac cgacaataaa tacgacgctg
      721 cgggatactc tgtagataat gaaaacccgc tctctggaaa agctggaggc gtggtcaaag
      781 tgacgtatcc aggactgacg aaggttctcg cactaaaagt ggataatgcc gaaactatta
      841 agaaagagtt aggtttaagt ctcactgaac cgttgatgga gcaagtcgga acggaagagt
      901 ttatcaaaag gttcggtgat ggtgcttcgc gtgtagtgct cagccttccc ttcgctgagg
      961 ggagttctag cgttgaatat attaataact gggaacaggc gaaagcgtta agcgtagaac
     1021 ttgagattaa ttttgaaacc cgtggaaaac gtggccaaga tgcgatgtat gagtatatgg
     1081 ctcaagcctg tgcaggaaat cgtgtcaggc gatctctttg tgaaggaacc ttacttctgt
     1141 ggtgtgacat aattggacaa actacctaca gagatttaaa gctctaaggt aaatataaaa
     1201 tttttaagtg tataatgtgt taaactactg attctaattg tttgtgtatt ttagattcca
     1261 acctatggaa ctgatgaatg ggagcagtgg tggaatgcag atcctagagc tcgctgatca
     1321 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1381 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     1441 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     1501 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     1561 gcggaaagaa ccagctgggg ctcgaggggg ggcccggtac cgagctcgaa ttcgtaatca
     1621 tgtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     1681 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     1741 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1801 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     1861 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     1921 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     1981 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2041 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2101 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2161 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2221 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2281 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2341 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacaag attagcagag
     2401 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2461 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2521 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     2581 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2641 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     2701 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     2761 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     2821 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     2881 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     2941 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3001 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     3061 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     3121 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3181 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     3241 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     3301 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     3361 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     3421 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     3481 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     3541 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     3601 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     3661 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3721 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     3781 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     3841 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     3901 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     3961 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     4021 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     4081 gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     4141 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     4201 ggttttccca gtcacgacgt tgtaaaacga cggccagtgc caagcttgat atctgaatta
     4261 tcaactttgt ataataaagt tgaacgagaa acgtaaaatg atataaatat caatatatta
     4321 aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca
     4381 ctatggcggc cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg
     4441 tgtggatttt gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga
     4501 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
     4561 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     4621 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     4681 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
     4741 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     4801 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     4861 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     4921 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     4981 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     5041 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     5101 gaatgcttaa tgaattacaa cagtactgcg atgagtggca ggcggggcgt aatctagagg
     5161 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     5221 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     5281 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     5341 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     5401 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     5461 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     5521 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     5581 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     5641 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     5701 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     5761 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     5821 agccagtctg caggtcgacc atagtgactg gatatgttgt gttttacagt attatgtagt
     5881 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
     5941 ttcaactttt ctatacaaag ttgatagctt gat
//
