LOCUS       sequence-305042-        7685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7685)
  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 7685)
  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-cjSTELLA-mCerulean-PuroTK
FEATURES             Location/Qualifiers
     source          1..7685
                     /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..93
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     regulatory      96..105
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             608..664
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(665..667,668..1378)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(707..728)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(968..987)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     repeat_region   1388..1450
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1621..1645
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        1652..2151
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1687..1710)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2084..2103
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2175..2771
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2175..2194)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2671..2691
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2778..3773
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     protein_bind    complement(4139..4163)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     repeat_region   4437..4471
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    complement(5020..5040)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(5101..5117)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5101..5117)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5114..5136)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5125..5141
                     /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(5149..5155,5156..5173,5174..5179))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5194..5215
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5332..5349)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5503..6091)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5583..5602)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6262..7053,7054..7122))
                     /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     6885..6904
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7123..7227)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7295..7313
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7351..7373)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7473..7492
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cccagtcacg acgttgtaaa acgacggcca gtgccaagct tgcatgcctg caggtcggga
       61 tatctgaatt atcaactttg tataataaag ttgttgccac catgggctta ccctaggggt
      121 cactcctagg tgttaggttg atactttttc acatgctgtt acaaacatct tcactgtaac
      181 ctgtgtagta agagaacaaa cacttcagca gcagaatgag ttgcatttaa ggcattcagg
      241 gaactgagac tagaacgttt tattgacaag ctgcacttgg taccactaca aagttactct
      301 caggctgcca ttctgtagta cccatatccc catcctcctt ttttttcctc ctaaaatgtt
      361 tttcttttgg ccccatgccc ttttttgtgt gttccctgtt ccaacattta cctaaacaaa
      421 cattatattt gagggttaat tacatttctc ttgggtacaa tattccaatt aattcatttc
      481 atcatttttt cagaatgcat caagaagagg aatgttcaaa tgtccctgca gtttctgcaa
      541 gtctagcgga tgggatcctt ctgagaatgc cagaatgggg aattacgaca ccaagccact
      601 tcagccagcc acgaacttct ctctgttaaa gcaagcagga gacgtggaag aaaaccccgg
      661 tcctgttagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      721 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
      781 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
      841 cctcgtgacc accctgacct ggggcgtgca gtgcttcgcc cgctaccccg accacatgaa
      901 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
      961 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1021 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1081 caagctggag tacaacgcca tcagcgacaa cgtctatatc accgccgaca agcagaagaa
     1141 cggcatcaag gccaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1201 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1261 ctacctgagc acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt
     1321 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga
     1381 attttaaccc tagaaagata atcatattgt gacgtacgtt aaagataatc atgcgtaaaa
     1441 ttgacgcatg tgttttatcg gtctgtatat cgaggtcccg tgaggcgttt gaatagatat
     1501 taagttttat tatatttaca cttacatact aataataaat tcaacaaaca attcttttac
     1561 gtgactttta agatttaact caaacaaaaa ctcaaaattt cttctataaa gtaacaaaac
     1621 tcaagtttgt acaaaaaagc agaattctac cgggtagggg aggcgctttt cccaaggcag
     1681 tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg
     1741 cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc
     1801 ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg
     1861 tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc
     1921 accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc
     1981 ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg
     2041 ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa
     2101 aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc gaccgatcat
     2161 caagcttgat cctcatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     2221 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     2281 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     2341 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     2401 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     2461 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     2521 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg
     2581 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     2641 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     2701 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     2761 agcccggtgc cggatccatg cccacgctac tgcgggttta tatagacggt ccccacggga
     2821 tggggaaaac caccaccacg caactgctgg tggccctggg ttcgcgcgac gatatcgtct
     2881 acgtacccga gccgatgact tactggcggg tgctgggggc ttccgagaca atcgcgaaca
     2941 tctacaccac acaacaccgc ctcgaccagg gtgagatatc ggccggggac gcggcggtgg
     3001 taatgacaag cgcccagata acaatgggca tgccttatgc cgtgaccgac gccgttctgg
     3061 ctcctcatat cgggggggag gctgggagct cacatgcccc gcccccggcc ctcaccctca
     3121 tcttcgaccg ccatcccatc gccgccctcc tgtgctaccc ggccgcgcgg taccttatgg
     3181 gcagcatgac cccccaggcc gtgctggcgt tcgtggccct catcccgccg accttgcccg
     3241 gcaccaacat cgtgcttggg gcccttccgg aggacagaca catcgaccgc ctggccaaac
     3301 gccagcgccc cggcgagcgg ctggacctgg ctatgctggc tgcgattcgc cgcgtttacg
     3361 ggctacttgc caatacggtg cggtatctgc agtgcggcgg gtcgtggcgg gaggactggg
     3421 gacagctttc ggggacggcc gtgccgcccc agggtgccga gccccagagc aacgcgggcc
     3481 cacgacccca tatcggggac acgttattta ccctgtttcg ggcccccgag ttgctggccc
     3541 ccaacggcga cctgtataac gtgtttgcct gggccttgga cgtcttggcc aaacgcctcc
     3601 gttccatgca cgtctttatc ctggattacg accaatcgcc cgccggctgc cgggacgccc
     3661 tgctgcaact tacctccggg atggtccaga cccacgtcac cacccccggc tccataccga
     3721 cgatatgcga cctggcgcgc acgtttgccc gggagatggg ggaggctaac tgagctctag
     3781 cttatcgact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     3841 agtcgagaaa ttgatgatct attaaacaat aaagatgtcc acatggaagt ttttcctgtc
     3901 atactttgtt aagaagggtg agaacagagt acctacattt tgaatggaag gattggagct
     3961 acgggggtgg gggtggggtg ggattagata aatgcctgct ctttactgaa ggctctttac
     4021 tattgcttta tgataatgtt tcatagttgg atatcataat ttaaacaagc aaaaccaaat
     4081 taagggccag ctcattcctc ccactcatga tctatagatc ctctagagtc gagggctgca
     4141 gcagctttct tgtacaaagt gggatatcta taacaagaaa atatatatat aataagttat
     4201 cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc ttaaaagtca
     4261 cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca aaatcagtga
     4321 cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg agatgtccta
     4381 aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca agaatgcatg
     4441 cgtcaatttt acgcagacta tctttctagg gttaataaac cttattcttg cccctttttt
     4501 ttcttgctgg taatttttta tagcagaccg agaaagctac tctctgctca tataaattat
     4561 atatcaataa ttttgataaa cgagttcaag gttgtatttt tcttgtatct ttttccctac
     4621 tatgatgcta gtaatttata agggatctgt gtagtttgaa tatatttgga taactttagc
     4681 tgtactgttt ggtttgtccc aaagaagcca agagcatgta aatattccca tgtgtttcag
     4741 aagcccaaag tcagtgaggt gaaacccagc aacaagacac tgaagcaaag atacttgtga
     4801 ataaagaaag cattagctag tttggttaga gcataataat tagattttct ggctttcaaa
     4861 aatttgggtt gcggtaagag caaagtttgt gctattttta cagttttcag tacaaaaagg
     4921 tgttaatata gaaacaataa agttgacatt agattacctt ttaaaaatag tttttgattt
     4981 tatagttgtt cttgctaaca attaggtgaa caatagtaac aacttttcta tacaaagttg
     5041 atagcttgat atcaatactc tagaggatcc ccgggtaccg agctcgaatt cgtaatcatg
     5101 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5161 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5221 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5281 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5341 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5401 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5461 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5521 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5581 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5641 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5701 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5761 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5821 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5881 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5941 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6001 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6061 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6121 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6181 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6241 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6301 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6361 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6421 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6481 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6541 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6601 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6661 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6721 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6781 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6841 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6901 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6961 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7021 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7081 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7141 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7201 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     7261 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     7321 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     7381 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     7441 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     7501 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     7561 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     7621 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     7681 gtttt
//
