LOCUS       sequence-301956-        4424 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 4424)
  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 4424)
  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-P5P4
FEATURES             Location/Qualifiers
     source          1..4424
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(347..652)
                     /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(449..468)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(972..1655)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     1570..1589
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1656..1758)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(1903..2134)
                     /gene="mutant version of attP"
                     /label=attP4
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2247..2266)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2258..3067
                     /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(2325..2344)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3210..3798
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3699..3718
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3933..3960)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4052..4138)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4192..4423
                     /gene="mutant version of attP"
                     /label=attP5
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 tcgaccgaca gccttccaaa tgttcttctc aaacggaatc gtcgtatcca gcctactcgc
       61 tattgtcctc aatgccgtat taaatcataa aaagaaataa gaaaaagagg tgcgagcctc
      121 ttttttgtgt gacaaaataa aaacatctac ctattcatat acgctagtgt catagtcctg
      181 aaaatcatct gcatcaagaa caatttcaca actcttatac ttttctctta caagtcgttc
      241 ggcttcatct ggattttcag cctctatact tactaaacgt gataaagttt ctgtaatttc
      301 tactgtatcg acctgcagac tggctgtgta taagggagcc tgacatttat attccccaga
      361 acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg gctgagatca gccacttctt
      421 ccccgataac ggagaccggc acactggcca tatcggtggt catcatgcgc cagctttcat
      481 ccccgatatg caccaccggg taaagttcac gggagacttt atctgacagc agacgtgcac
      541 tggccagggg gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc tgtacatcca
      601 caaacagacg ataacggctc tctcttttat aggtgtaaac cttaaactgc atttcaccag
      661 cccctgttct cgtcagcaaa agagccgttc atttcaataa accgggcgac ctcagccatc
      721 ccttcctgat tttccgcttt ccagcgttcg gcacgcagac gacgggcttc attctgcatg
      781 gttgtgctta ccagaccgga gatattgaca tcatatatgc cttgagcaac tgatagctgt
      841 cgctgtcaac tgtcactgta atacgctgct tcatagcata cctctttttg acatacttcg
      901 ggtatacata tcagtatata ttcttatacc gcaaaaatca gcgcgcaaat acgcatactg
      961 ttatctggct tttagtaagc cggatccacg cggcgtttac gccccccctg ccactcatcg
     1021 cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga
     1081 tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg
     1141 gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa
     1201 ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag
     1261 gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa
     1321 tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg
     1381 taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat
     1441 tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc
     1501 ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag
     1561 gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata
     1621 tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat
     1681 ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa
     1741 cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg cttcccggta
     1801 tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac aggtatttat
     1861 tcggcgcaaa gtgcgtcggg tgatgctgcc aacttagtcg actacaggtc actaatacca
     1921 tctaagtagt tgattcatag tgactggata tgttgtgttt tacagtatta tgtagtctgt
     1981 tttttatgca aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca
     2041 acttttctat acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa
     2101 caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgcagc tctggcccgt
     2161 gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa
     2221 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     2281 gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
     2341 cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc
     2401 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     2461 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     2521 tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat tccaggtatt
     2581 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     2641 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     2701 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     2761 taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc
     2821 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     2881 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     2941 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     3001 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     3061 tttctaatca gaattggtta attggttgta acactggcag agcattacgc tgacttgacg
     3121 ggacggcgca agctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3181 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3241 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3301 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3361 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3421 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3481 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3541 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3601 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3661 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3721 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3781 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3841 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3901 accgctagcc aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct
     3961 gcttagtttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt
     4021 gcttcacaac gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg
     4081 acaaacaaca gataaaacga aaggcccagt cttccgactg agcctttcgt tttatttgat
     4141 gcctggcagt tccctactct cgcgttaacg ctagcatgga tctcgggccc caaataatga
     4201 ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa tgctttttta
     4261 taatgccaac tttgtataca aaagttgaac gagaaacgta aaatgatata aatatcaata
     4321 tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca caacatatcc
     4381 agtcactatg aatcaactac ttagatggta ttagtgacct gtag
//
