LOCUS       sequence-304882-        4419 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 4419)
  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 4419)
  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-R3R4(R)
FEATURES             Location/Qualifiers
     source          1..4419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..23
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     15..32
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     16..32
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    73..196
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(221..226,227..244,245..251)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             305..985
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(371..390)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1305..1610
                     /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     1489..1508
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1651..1774)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1835..1851)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1835..1851)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1848..1870)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1859..1875
                     /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(1883..1889,1890..1907,1908..1913))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1928..1949
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2066..2083)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2237..2825)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2317..2336)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2996..3787,3788..3856))
                     /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     3619..3638
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3857..3961)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4029..4047
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4085..4107)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4207..4226
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cccagtcacg acgttgtaaa acgacggcca gtgccaagct tgcatgcctg caggtcggga
       61 tatctgaatt atcaactttg tataataaag ttgaacgaga aacgtaaaat gatataaata
      121 tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac
      181 atatccagtc actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg
      241 ctcgtataat gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc
      301 taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa
      361 agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct
      421 ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt
      481 tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga
      541 cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac
      601 tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat
      661 atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat
      721 tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa
      781 cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca
      841 aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt
      901 ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc aggcggggcg
      961 taatctagag gatccggctt actaaaagcc agataacagt atgcgtattt gcgcgctgat
     1021 ttttgcggta taagaatata tactgatatg tatacccgaa gtatgtcaaa aagaggtatg
     1081 ctatgaagca gcgtattaca gtgacagttg acagcgacag ctatcagttg ctcaaggcat
     1141 atatgatgtc aatatctccg gtctggtaag cacaaccatg cagaatgaag cccgtcgtct
     1201 gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg gctgaggtcg cccggtttat
     1261 tgaaatgaac ggctcttttg ctgacgagaa caggggctgg tgaaatgcag tttaaggttt
     1321 acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg
     1381 acacgcccgg gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag
     1441 tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca
     1501 ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat ctcagccacc
     1561 gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa atgtcaggct
     1621 cccttataca cagccagtct gcaggtcgac catagtgact ggatatgttg tgttttacag
     1681 tattatgtag tctgtttttt atgcaaaatc taatttaata tattgatatt tatatcattt
     1741 tacgtttctc gttcaacttt tctatacaaa gttgatagct tgatatcaat actctagagg
     1801 atccccgggt accgagctcg aattcgtaat catggtcata gctgtttcct gtgtgaaatt
     1861 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     1921 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1981 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2041 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2101 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2161 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2221 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2281 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2341 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2401 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2461 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2521 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2581 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2641 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2701 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2761 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2821 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2881 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2941 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3001 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3061 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3121 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3181 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3241 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3301 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3361 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3421 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3481 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3541 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3601 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3661 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3721 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3781 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3841 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3901 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3961 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     4021 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     4081 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     4141 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     4201 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     4261 acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact
     4321 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     4381 gtgctgcaag gcgattaagt tgggtaacgc cagggtttt
//
