LOCUS       sequence-301953-        4795 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 4795)
  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 4795)
  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: pDONR-P3P1r
FEATURES             Location/Qualifiers
     source          1..4795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..234
                     /gene="mutant version of attP"
                     /label=attP3
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(630..935)
                     /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     complement(732..751)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1255..1938)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     1853..1872
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1939..2041)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    2199..2430
                     /gene="mutant version of attP"
                     /label=attP1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2469..2488)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2470..2488)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2493..2509)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2493..2509)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2611..2630)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2622..3431
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2689..2708)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3578..4166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4067..4086
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4320..4337
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4496..4523)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4615..4701)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4750..4772
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4764..4781
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4765..4781
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ccaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca
       61 atgctttttt ataatgccaa ctttgtataa taaagttgaa cgagaaacgt aaaatgatat
      121 aaatatcaat atattaaatt agattttgca taaaaaacag actacataat actgtaaaac
      181 acaacatatc cagtcactat gaatcaacta cttagatggt attagtgacc tgtagtcgac
      241 cgacagcctt ccaaatgttc ttcgggtgat gctgccaact tagtcgaccg acagccttcc
      301 aaatgttctt ctcaaacgga atcgtcgtat ccagcctact cgctattgtc ctcaatgccg
      361 tattaaatca taaaaagaaa taagaaaaag aggtgcgagc ctcttttttg tgtgacaaaa
      421 taaaaacatc tacctattca tatacgctag tgtcatagtc ctgaaaatca tctgcatcaa
      481 gaacaatttc acaactctta tacttttctc ttacaagtcg ttcggcttca tctggatttt
      541 cagcctctat acttactaaa cgtgataaag tttctgtaat ttctactgta tcgacctgca
      601 gactggctgt gtataaggga gcctgacatt tatattcccc agaacatcag gttaatggcg
      661 tttttgatgt cattttcgcg gtggctgaga tcagccactt cttccccgat aacggagacc
      721 ggcacactgg ccatatcggt ggtcatcatg cgccagcttt catccccgat atgcaccacc
      781 gggtaaagtt cacgggagac tttatctgac agcagacgtg cactggccag ggggatcacc
      841 atccgtcgcc cgggcgtgtc aataatatca ctctgtacat ccacaaacag acgataacgg
      901 ctctctcttt tataggtgta aaccttaaac tgcatttcac cagcccctgt tctcgtcagc
      961 aaaagagccg ttcatttcaa taaaccgggc gacctcagcc atcccttcct gattttccgc
     1021 tttccagcgt tcggcacgca gacgacgggc ttcattctgc atggttgtgc ttaccagacc
     1081 ggagatattg acatcatata tgccttgagc aactgatagc tgtcgctgtc aactgtcact
     1141 gtaatacgct gcttcatagc atacctcttt ttgacatact tcgggtatac atatcagtat
     1201 atattcttat accgcaaaaa tcagcgcgca aatacgcata ctgttatctg gcttttagta
     1261 agccggatcc acgcggcgtt tacgcccccc ctgccactca tcgcagtact gttgtaattc
     1321 attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag
     1381 cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa
     1441 gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc
     1501 tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta
     1561 acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact
     1621 ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact
     1681 atcccatatc accagctcac cgtctttcat tgccatacgg aattccggat gagcattcat
     1741 caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt
     1801 ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga
     1861 ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc
     1921 agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa
     1981 tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc
     2041 aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg
     2101 atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc
     2161 gggtgatgct gccaacttag tcgacgctct agagctccaa ataatgattt tattttgact
     2221 gatagtgacc tgttcgttgc aacaaattga taagcaatgc ttttttataa tgccaacttt
     2281 gtacaaaaaa gttgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat
     2341 tttgcataaa aaacagacta cataatactg taaaacacaa catatgcagt cactatgaac
     2401 caactactta gatggtatta gtgacctgta gaattcgagc tctagagctg cagggcggcc
     2461 gcgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc
     2521 ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa
     2581 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     2641 aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     2701 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg
     2761 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     2821 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     2881 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg
     2941 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     3001 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     3061 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     3121 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
     3181 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     3241 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     3301 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     3361 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     3421 agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt
     3481 gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg tcgttccact
     3541 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3601 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3661 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3721 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3781 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3841 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3901 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3961 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4021 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4081 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4141 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4201 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4261 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4321 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     4381 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     4441 agcgcaacgc aattaatacg cgtaccgcta gccaggaaga gtttgtagaa acgcaaaaag
     4501 gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc
     4561 tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg gcggatttgt
     4621 cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtcttccga
     4681 ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgtta acgctagcat
     4741 ggatgttttc ccagtcacga cgttgtaaaa cgacggccag tcttaagctc gggcc
//
