LOCUS       sequence-304970-        4959 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 4959)
  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 4959)
  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-DEST-R5R4
FEATURES             Location/Qualifiers
     source          1..4959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    24..147
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(576..881)
                     /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(678..697)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1201..1884)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     1799..1818
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1885..1987)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2165..2288)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2317..2334)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2317..2333)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2326..2348)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2542..2561)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2661..2683
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2721..2739)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2807..2911
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2912..2980,2981..3772)
                     /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(3130..3149)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3943..4531
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4432..4451
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4685..4702
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4819..4840
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4855..4860,4861..4878,4879..4885)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4893..4909
                     /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)."
     primer_bind     4898..4920
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4917..4933
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4917..4933
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ccggggatcc tctagagtcg gggcaacttt gtatacaaaa gttgaacgag aaacgtaaaa
       61 tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta cataatactg
      121 taaaacacaa catatccagt cactatgaat caactactta gatggtatta gtgacctgta
      181 gtcgaccgac agccttccaa atgttcttcg ggtgatgctg ccaacttagt cgaccgacag
      241 ccttccaaat gttcttctca aacggaatcg tcgtatccag cctactcgct attgtcctca
      301 atgccgtatt aaatcataaa aagaaataag aaaaagaggt gcgagcctct tttttgtgtg
      361 acaaaataaa aacatctacc tattcatata cgctagtgtc atagtcctga aaatcatctg
      421 catcaagaac aatttcacaa ctcttatact tttctcttac aagtcgttcg gcttcatctg
      481 gattttcagc ctctatactt actaaacgtg ataaagtttc tgtaatttct actgtatcga
      541 cctgcagact ggctgtgtat aagggagcct gacatttata ttccccagaa catcaggtta
      601 atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc cccgataacg
      661 gagaccggca cactggccat atcggtggtc atcatgcgcc agctttcatc cccgatatgc
      721 accaccgggt aaagttcacg ggagacttta tctgacagca gacgtgcact ggccaggggg
      781 atcaccatcc gtcgcccggg cgtgtcaata atatcactct gtacatccac aaacagacga
      841 taacggctct ctcttttata ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc
      901 gtcagcaaaa gagccgttca tttcaataaa ccgggcgacc tcagccatcc cttcctgatt
      961 ttccgctttc cagcgttcgg cacgcagacg acgggcttca ttctgcatgg ttgtgcttac
     1021 cagaccggag atattgacat catatatgcc ttgagcaact gatagctgtc gctgtcaact
     1081 gtcactgtaa tacgctgctt catagcatac ctctttttga catacttcgg gtatacatat
     1141 cagtatatat tcttataccg caaaaatcag cgcgcaaata cgcatactgt tatctggctt
     1201 ttagtaagcc ggatccacgc ggcgtttacg ccccccctgc cactcatcgc agtactgttg
     1261 taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat
     1321 cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg
     1381 ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg
     1441 attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc
     1501 accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta
     1561 ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg
     1621 aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc
     1681 attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt
     1741 tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc
     1801 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
     1861 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc
     1921 aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg
     1981 ccgatcaacg tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac
     2041 accaggattt atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag
     2101 tgcgtcgggt gatgctgcca acttagtcga ctacaggtca ctaataccat ctaagtagtt
     2161 gattcatagt gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa
     2221 aatctaattt aatatattga tatttatatc attttacgtt tctcgttcaa cttttctata
     2281 caaagttgcc ccgacctgca ggcatgcaag cttggcactg gccgtcgttt tacaacgtcg
     2341 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     2401 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     2461 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2521 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     2581 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     2641 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2701 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     2761 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2821 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2881 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     2941 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3001 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3061 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3121 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     3181 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3241 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3301 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3361 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3421 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     3481 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     3541 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3601 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     3661 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     3721 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     3781 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     3841 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     3901 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3961 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4021 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4081 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4141 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4201 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4261 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4321 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4381 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4441 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4501 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4561 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4621 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4681 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     4741 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     4801 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     4861 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     4921 aaacagctat gaccatgatt acgaattcga gctcggtac
//
