LOCUS       sequence-305023-        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: pUC-DEST-R1R2
FEATURES             Location/Qualifiers
     source          1..4458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    48..172
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(197..202,203..220,221..227)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             281..940
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(347..366)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1282..1587
                     /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     1466..1485
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1628..1752)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1816..1833)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1816..1832)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1825..1847)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2041..2060)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2160..2182
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2220..2238)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2306..2410
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2411..2479,2480..3271)
                     /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(2629..2648)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3442..4030
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3931..3950
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4184..4201
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4318..4339
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4354..4359,4360..4377,4378..4384)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4392..4408
                     /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     4397..4419
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4416..4432
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4416..4432
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cgagctcgga tccactagtc cagtgtggtg gaattctgca gatatcaaca agtttgtaca
       61 aaaaagctga acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc
      121 ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta tggcggccgc
      181 attaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt ggattttgag
      241 ttaggatccg tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg
      301 atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc
      361 agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac
      421 cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat
      481 gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag
      541 tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag
      601 tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta
      661 cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc
      721 caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt
      781 cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct
      841 ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga
      901 attacaacag tactgcgatg agtggcaggg cggggcgtaa agatctggat ccggcttact
      961 aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa gaatatatac
     1021 tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg
     1081 acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat atctccggtc
     1141 tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg
     1201 aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc tcttttgctg
     1261 acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag agagagccgt
     1321 tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg
     1381 atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg
     1441 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc
     1501 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc
     1561 attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag ccagtctgca
     1621 ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct gttttttatg
     1681 caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt cagctttctt
     1741 gtacaaagtg gttgatatcc agcacagtgg cggccgctcg agtctagagt cgacctgcag
     1801 gcatgcaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     1861 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     1921 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     1981 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     2041 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     2101 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     2161 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     2221 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     2281 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2341 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2401 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2461 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2521 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2581 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2641 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2701 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2761 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2821 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     2881 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     2941 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3001 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3061 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3121 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3181 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3241 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3301 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3361 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3421 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3481 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3541 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3601 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3661 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3721 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3781 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3841 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3901 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3961 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4021 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4081 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4141 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4201 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     4261 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     4321 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     4381 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     4441 cgaattcgag ctcggtac
//
