LOCUS       sequence-304934-        6918 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 6918)
  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 6918)
  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: pPB-DEST-R1R2-pNeo
FEATURES             Location/Qualifiers
     source          1..6918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   660..694
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1017..1141
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1166..1171,1172..1189,1190..1196)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1250..1909
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1316..1335)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2251..2556
                     /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     2435..2454
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2597..2721)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2914..3413
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2949..2972)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3346..3365
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3427..4230
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3490..3509)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4100..4119
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     repeat_region   4644..4706
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(4730..4748)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4769..4785)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4769..4785)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4782..4804)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4793..4809
                     /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(4817..4823,4824..4841,4842..4847))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4862..4883
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5000..5017)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5171..5759)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5251..5270)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5930..6721,6722..6790))
                     /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     6553..6572
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6791..6895)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccttaac
      661 cctagaaaga tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt
      721 ctaaatagcg cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc
      781 cgtgaggcgt gcttgtcaat gaggtaagtg tcactgattt tgaactataa cgaccgcgtg
      841 agtcaaaatg acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat
      901 tatattgtta tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta
      961 tagatgatcc actagtaacg gccgccagtg tgctggaatt cgcggccgct agcatcacaa
     1021 gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt
     1081 agattttgca taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat
     1141 ggcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg
     1201 gattttgagt taggatccgt cgagattttc aggagctaag gaagctaaaa tggagaaaaa
     1261 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
     1321 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
     1381 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
     1441 ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat
     1501 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
     1561 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
     1621 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     1681 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     1741 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct
     1801 gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat
     1861 gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa cgcgtggatc
     1921 cggcttacta aaagccagat aacagtatgc gtatttgcgc gctgattttt gcggtataag
     1981 aatatatact gatatgtata cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt
     2041 attacagtga cagttgacag cgacagctat cagttgctca aggcatatat gatgtcaata
     2101 tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt gccgaacgct
     2161 ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa atgaacggct
     2221 cttttgctga cgagaacagg ggctggtgaa atgcagttta aggtttacac ctataaaaga
     2281 gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga
     2341 cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt
     2401 tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt
     2461 gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc
     2521 aaaaacgcca ttaacctgat gttctgggga atataaatgt caggctccct tatacacagc
     2581 cagtctgcag gtcgaccata gtgactggat atgttgtgtt ttacagtatt atgtagtctg
     2641 ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg tttctcgttc
     2701 agctttcttg tacaaagtgg tgatgctacc ggaacatcga gggatcaagc tttaatgcgg
     2761 tagtttatca cagttaaatt gctaacgcag tcaggcaccg tgtatgaaat ctaacaatgc
     2821 gctcatcgtc atcctcggca ccgtcaccct ggatgctgta ggcataggct tggttatgcc
     2881 ggtactgccg ggcctcttgc ggggaattct accgggtagg ggaggcgctt ttcccaaggc
     2941 agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct
     3001 ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc
     3061 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
     3121 cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca
     3181 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
     3241 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
     3301 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     3361 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca
     3421 gccaatatgg gatcggccat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3481 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3541 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3601 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3661 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3721 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3781 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3841 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3901 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3961 gcgcgcatgc ccgacggcga tgatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     4021 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     4081 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4141 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4201 ttctatcgcc ttcttgacga gttcttctga ggggatcgat ccgctgtaag tctgcagaaa
     4261 ttgatgatct attaaacaat aaagatgtcc actaaaatgg aagtttttcc tgtcatactt
     4321 tgttaagaag ggtgagaaca gagtacctac attttgaatg gaaggattgg agctacgggg
     4381 gtgggggtgg ggtgggatta gataaatgcc tgctctttac tgaaggctct ttactattgc
     4441 tttatgataa tgtttcatag ttggatatca gatcctttgt tactttatag aagaaatttt
     4501 gagtttttgt ttttttttaa taaataaata aacataaata aattgtttgt tgaatttatt
     4561 attagtatgt aagtgtaaat ataataaaac ttaatatcta ttcaaattaa taaataaacc
     4621 tcgatataca gaccgataaa acacatgcgt caattttacg catgattatc tttaacgtac
     4681 gtcacaatat gattatcttt ctagggttaa agagctccag cttttgttcc ctttagtgag
     4741 ggttaatttc gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4801 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4861 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4921 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4981 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5041 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5101 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5161 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5221 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5281 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5341 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5401 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5461 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5521 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5581 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5641 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5701 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5761 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5821 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5881 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5941 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6001 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6061 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6121 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6181 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6241 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6301 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6361 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6421 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6481 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6541 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6601 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6661 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6721 gagcaaaaac gggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6781 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6841 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6901 ttccccgaaa agtgccac
//
