LOCUS       sequence-305021-        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-R1R4
FEATURES             Location/Qualifiers
     source          1..4959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(25..42)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(25..41)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(34..56)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(250..269)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     369..391
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(429..447)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        515..619
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(620..688,689..1480)
                     /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(838..857)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1651..2239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2140..2159
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2393..2410
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2527..2548
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2563..2568,2569..2586,2587..2593)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2601..2617
                     /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     2606..2628
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2625..2641
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2625..2641
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    2691..2814
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(3243..3548)
                     /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(3345..3364)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3868..4551)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     4466..4485
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4552..4654)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(4832..4955)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gacctgcagg catgcaagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa
       61 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
      121 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
      181 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
      241 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
      301 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
      361 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
      421 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
      481 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
      541 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
      601 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
      661 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
      721 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
      781 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
      841 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
      901 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
      961 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     1021 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     1081 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     1141 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     1201 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     1261 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     1321 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     1381 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     1441 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     1501 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     1561 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1621 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1681 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1741 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     1801 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     1861 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1921 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     1981 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     2041 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     2101 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     2161 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     2221 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     2281 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     2341 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     2401 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     2461 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     2521 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     2581 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     2641 ccatgattac gaattcgagc tcggtacccg gggatcctct agagtcgggg caagtttgta
     2701 caaaaaagct gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt
     2761 gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatgaatcaa
     2821 ctacttagat ggtattagtg acctgtagtc gaccgacagc cttccaaatg ttcttcgggt
     2881 gatgctgcca acttagtcga ccgacagcct tccaaatgtt cttctcaaac ggaatcgtcg
     2941 tatccagcct actcgctatt gtcctcaatg ccgtattaaa tcataaaaag aaataagaaa
     3001 aagaggtgcg agcctctttt ttgtgtgaca aaataaaaac atctacctat tcatatacgc
     3061 tagtgtcata gtcctgaaaa tcatctgcat caagaacaat ttcacaactc ttatactttt
     3121 ctcttacaag tcgttcggct tcatctggat tttcagcctc tatacttact aaacgtgata
     3181 aagtttctgt aatttctact gtatcgacct gcagactggc tgtgtataag ggagcctgac
     3241 atttatattc cccagaacat caggttaatg gcgtttttga tgtcattttc gcggtggctg
     3301 agatcagcca cttcttcccc gataacggag accggcacac tggccatatc ggtggtcatc
     3361 atgcgccagc tttcatcccc gatatgcacc accgggtaaa gttcacggga gactttatct
     3421 gacagcagac gtgcactggc cagggggatc accatccgtc gcccgggcgt gtcaataata
     3481 tcactctgta catccacaaa cagacgataa cggctctctc ttttataggt gtaaacctta
     3541 aactgcattt caccagcccc tgttctcgtc agcaaaagag ccgttcattt caataaaccg
     3601 ggcgacctca gccatccctt cctgattttc cgctttccag cgttcggcac gcagacgacg
     3661 ggcttcattc tgcatggttg tgcttaccag accggagata ttgacatcat atatgccttg
     3721 agcaactgat agctgtcgct gtcaactgtc actgtaatac gctgcttcat agcatacctc
     3781 tttttgacat acttcgggta tacatatcag tatatattct tataccgcaa aaatcagcgc
     3841 gcaaatacgc atactgttat ctggctttta gtaagccgga tccacgcggc gtttacgccc
     3901 cccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc
     3961 atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt
     4021 ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa
     4081 atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa
     4141 ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg
     4201 tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg
     4261 ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt
     4321 cattgccata cggaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc
     4381 cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg
     4441 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg
     4501 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc
     4561 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt
     4621 atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc aaaagttggc
     4681 ccagggcttc ccggtatcaa cagggacacc aggatttatt tattctgcga agtgatcttc
     4741 cgtcacaggt atttattcgg cgcaaagtgc gtcgggtgat gctgccaact tagtcgacta
     4801 caggtcacta ataccatcta agtagttgat tcatagtgac tggatatgtt gtgttttaca
     4861 gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt
     4921 ttacgtttct cgttcaactt ttctatacaa agttgcccc
//
