LOCUS       sequence-301954-        4458 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 4458)
  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 4458)
  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-P2rP4
FEATURES             Location/Qualifiers
     source          1..4458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(24..255)
                     /gene="mutant version of attP"
                     /label=attP2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(616..921)
                     /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(718..737)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1241..1924)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     1839..1858
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1925..2027)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2172..2403)
                     /gene="mutant version of attP"
                     /label=attP4
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2516..2535)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2527..3336
                     /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(2594..2613)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3479..4067
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3968..3987
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4202..4229)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4321..4407)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctgcagctct agagctcgaa ttctacaggt cactaatacc atctaagtag ttggttcata
       61 gtgactgcat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat
      121 ttaatatatt gatatttata tcattttacg tttctcgttc aactttcttg tacaaagttg
      181 gcattataaa aaagcattgc ttatcaattt gttgcaacga acaggtcact atcagtcaaa
      241 ataaaatcat tatttggagc tctagagcgt cgaccgacag ccttccaaat gttcttctca
      301 aacggaatcg tcgtatccag cctactcgct attgtcctca atgccgtatt aaatcataaa
      361 aagaaataag aaaaagaggt gcgagcctct tttttgtgtg acaaaataaa aacatctacc
      421 tattcatata cgctagtgtc atagtcctga aaatcatctg catcaagaac aatttcacaa
      481 ctcttatact tttctcttac aagtcgttcg gcttcatctg gattttcagc ctctatactt
      541 actaaacgtg ataaagtttc tgtaatttct actgtatcga cctgcagact ggctgtgtat
      601 aagggagcct gacatttata ttccccagaa catcaggtta atggcgtttt tgatgtcatt
      661 ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gagaccggca cactggccat
      721 atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt aaagttcacg
      781 ggagacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc gtcgcccggg
      841 cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct ctcttttata
      901 ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc gtcagcaaaa gagccgttca
      961 tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc cagcgttcgg
     1021 cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cagaccggag atattgacat
     1081 catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa tacgctgctt
     1141 catagcatac ctctttttga catacttcgg gtatacatat cagtatatat tcttataccg
     1201 caaaaatcag cgcgcaaata cgcatactgt tatctggctt ttagtaagcc ggatccacgc
     1261 ggcgtttacg ccccccctgc cactcatcgc agtactgttg taattcatta agcattctgc
     1321 cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc atcagcacct
     1381 tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat
     1441 tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag acgaaaaaca
     1501 tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac gccacatctt
     1561 gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag agcgatgaaa
     1621 acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc catatcacca
     1681 gctcaccgtc tttcattgcc atacggaatt ccggatgagc attcatcagg cgggcaagaa
     1741 tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt aaaaaggccg
     1801 taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa
     1861 aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg atttttttct
     1921 ccattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg
     1981 atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaacg tctcattttc
     2041 gccaaaagtt ggcccagggc ttcccggtat caacagggac accaggattt atttattctg
     2101 cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt gatgctgcca
     2161 acttagtcga ctacaggtca ctaataccat ctaagtagtt gattcatagt gactggatat
     2221 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
     2281 tatttatatc attttacgtt tctcgttcaa cttttctata caaagttggc attataagaa
     2341 agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta
     2401 tttgccatcc agctgcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa
     2461 gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     2521 ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat
     2581 gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc
     2641 tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc
     2701 gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct
     2761 cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg
     2821 atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt
     2881 gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct
     2941 tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg
     3001 gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa
     3061 gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca
     3121 cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc
     3181 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct
     3241 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa
     3301 ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa
     3361 cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc aaaatccctt
     3421 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3481 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3541 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3601 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     3661 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3721 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3781 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3841 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3901 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3961 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4021 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4081 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4141 tatcccctga ttctgtggat aaccgtatta ccgctagcca ggaagagttt gtagaaacgc
     4201 aaaaaggcca tccgtcagga tggccttctg cttagtttga tgcctggcag tttatggcgg
     4261 gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg ttcaaatccg ctcccggcgg
     4321 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
     4381 ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcgttaacgc
     4441 tagcatggat ctcgggcc
//
