LOCUS       sequence-304977-        7476 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 7476)
  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 7476)
  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-R5R4-pNeo
FEATURES             Location/Qualifiers
     source          1..7476
                     /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    1021..1144
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1573..1878)
                     /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(1675..1694)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2198..2881)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2796..2815
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2882..2984)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(3162..3285)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3472..3971
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3507..3530)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3904..3923
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3985..4788
                     /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(4048..4067)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4658..4677
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     repeat_region   5202..5264
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(5288..5306)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5327..5343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5327..5343)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5340..5362)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5351..5367
                     /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(5375..5381,5382..5399,5400..5405))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5420..5441
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5558..5575)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5729..6317)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5809..5828)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6488..7279,7280..7348))
                     /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     7111..7130
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7349..7453)
                     /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 agagtcgggg
     1021 caactttgta tacaaaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa
     1081 attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac
     1141 tatgaatcaa ctacttagat ggtattagtg acctgtagtc gaccgacagc cttccaaatg
     1201 ttcttcgggt gatgctgcca acttagtcga ccgacagcct tccaaatgtt cttctcaaac
     1261 ggaatcgtcg tatccagcct actcgctatt gtcctcaatg ccgtattaaa tcataaaaag
     1321 aaataagaaa aagaggtgcg agcctctttt ttgtgtgaca aaataaaaac atctacctat
     1381 tcatatacgc tagtgtcata gtcctgaaaa tcatctgcat caagaacaat ttcacaactc
     1441 ttatactttt ctcttacaag tcgttcggct tcatctggat tttcagcctc tatacttact
     1501 aaacgtgata aagtttctgt aatttctact gtatcgacct gcagactggc tgtgtataag
     1561 ggagcctgac atttatattc cccagaacat caggttaatg gcgtttttga tgtcattttc
     1621 gcggtggctg agatcagcca cttcttcccc gataacggag accggcacac tggccatatc
     1681 ggtggtcatc atgcgccagc tttcatcccc gatatgcacc accgggtaaa gttcacggga
     1741 gactttatct gacagcagac gtgcactggc cagggggatc accatccgtc gcccgggcgt
     1801 gtcaataata tcactctgta catccacaaa cagacgataa cggctctctc ttttataggt
     1861 gtaaacctta aactgcattt caccagcccc tgttctcgtc agcaaaagag ccgttcattt
     1921 caataaaccg ggcgacctca gccatccctt cctgattttc cgctttccag cgttcggcac
     1981 gcagacgacg ggcttcattc tgcatggttg tgcttaccag accggagata ttgacatcat
     2041 atatgccttg agcaactgat agctgtcgct gtcaactgtc actgtaatac gctgcttcat
     2101 agcatacctc tttttgacat acttcgggta tacatatcag tatatattct tataccgcaa
     2161 aaatcagcgc gcaaatacgc atactgttat ctggctttta gtaagccgga tccacgcggc
     2221 gtttacgccc cccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga
     2281 catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt
     2341 cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg
     2401 ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat
     2461 tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg
     2521 aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg
     2581 tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct
     2641 caccgtcttt cattgccata cggaattccg gatgagcatt catcaggcgg gcaagaatgt
     2701 gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa
     2761 tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat
     2821 gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca
     2881 ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc
     2941 ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc
     3001 aaaagttggc ccagggcttc ccggtatcaa cagggacacc aggatttatt tattctgcga
     3061 agtgatcttc cgtcacaggt atttattcgg cgcaaagtgc gtcgggtgat gctgccaact
     3121 tagtcgacta caggtcacta ataccatcta agtagttgat tcatagtgac tggatatgtt
     3181 gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat
     3241 ttatatcatt ttacgtttct cgttcaactt ttctatacaa agttgccccg acctgcaggc
     3301 atgcaagctt taatgcggta gtttatcaca gttaaattgc taacgcagtc aggcaccgtg
     3361 tatgaaatct aacaatgcgc tcatcgtcat cctcggcacc gtcaccctgg atgctgtagg
     3421 cataggcttg gttatgccgg tactgccggg cctcttgcgg ggaattctac cgggtagggg
     3481 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     3541 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg
     3601 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     3661 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta
     3721 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     3781 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     3841 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     3901 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     3961 cgggcctttc gacctgcagc caatatggga tcggccattg aacaagatgg attgcacgca
     4021 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     4081 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     4141 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     4201 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     4261 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4321 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     4381 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4441 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     4501 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgatg atctcgtcgt gacccatggc
     4561 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4621 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4681 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4741 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagg ggatcgatcc
     4801 gctgtaagtc tgcagaaatt gatgatctat taaacaataa agatgtccac taaaatggaa
     4861 gtttttcctg tcatactttg ttaagaaggg tgagaacaga gtacctacat tttgaatgga
     4921 aggattggag ctacgggggt gggggtgggg tgggattaga taaatgcctg ctctttactg
     4981 aaggctcttt actattgctt tatgataatg tttcatagtt ggatatcaga tcctttgtta
     5041 ctttatagaa gaaattttga gtttttgttt ttttttaata aataaataaa cataaataaa
     5101 ttgtttgttg aatttattat tagtatgtaa gtgtaaatat aataaaactt aatatctatt
     5161 caaattaata aataaacctc gatatacaga ccgataaaac acatgcgtca attttacgca
     5221 tgattatctt taacgtacgt cacaatatga ttatctttct agggttaaag agctccagct
     5281 tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc
     5341 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5401 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5461 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5521 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5581 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5641 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5701 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5761 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5821 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5881 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5941 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6001 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6061 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6121 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6181 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6241 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6301 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6361 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6421 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6481 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6541 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6601 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6661 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6721 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6781 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6841 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6901 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6961 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7021 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7081 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7141 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7201 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7261 tcaccagcgt ttctgggtga gcaaaaacgg gaaggcaaaa tgccgcaaaa aagggaataa
     7321 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7381 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7441 taggggttcc gcgcacattt ccccgaaaag tgccac
//
