LOCUS       sequence-103222-        3246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for mCFP knock-in via landing pad.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3246)
  AUTHORS   Ohtsuka M, Miura H, Mochida K, Hirose M, Hasegawa A, Ogura A, 
            Mizutani R, Kimura M, Isotani A, Ikawa M, Sato M, Gurumurthy CB
  TITLE     One-step generation of multiple transgenic mouse lines using an 
            improved Pronuclear Injection-based Targeted Transgenesis (i-PITT).
  JOURNAL   BMC Genomics. 2015 Apr 9;16:274. doi: 10.1186/s12864-015-1432-5.
  PUBMED    25887549
REFERENCE   2  (bases 1 to 3246)
  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: "BMC 
            Genomics."; date: "2015-04-9"; volume: "16:274. doi"; pages: " 
            10.1186/s12864-015-1432-5"
COMMENT     Sequence Label: pBGV
FEATURES             Location/Qualifiers
     source          1..3246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..22
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6..22
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    161..194
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     protein_bind    complement(203..236)
                     /label=loxJTZ17
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      293..302
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(299..301,302..304,305..1018)
                     /codon_start=1
                     /product="monomeric super enhanced variant of CFP (Matsuda 
                     et al., 2008)"
                     /label=mseCFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             join(299..301,302..304,305..1018)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             join(299..301,302..304,305..1018)
                     /codon_start=1
                     /product="enhanced CFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(344..365)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(605..624)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     952..973
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1145..1266
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1182..1201)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1236..1255
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1382..1429)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    complement(1430..1463)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(1547..1563)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1547..1563)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      1611..1642
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        1643..1745
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1746..2561
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1819..1838)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2585..2612
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(2624..3211)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2704..2723)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cccgtgtaaa acgacggcca gtttatctag tcagcttgat tctagctgat cgtggaccgg
       61 aaggtgagcc agtgagttga ttgcagtcca gttacgctgg agtctgaggc tcgtcctgaa
      121 tgatatgcgg cctcggatcc gcgatcgatc cgcggtgcgg gtgccagggc gtgcccttgg
      181 gctccccggg cgcgtactcc acataacttc gtatagcata cattatagca atttatcgaa
      241 gttcctattc cgaagttcct attctatcag aagtatagga acttcaccgg tcgccaccat
      301 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      361 cgacgtaaac ggccacaggt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
      421 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
      481 cgtgaccacc ctgacctggg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
      541 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgta ccatcttctt
      601 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
      661 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
      721 gctggagtac aactacatca gccacaacgt ctatatcacc gccgacaagc agaagaacgg
      781 catcaaggcc cacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
      841 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
      901 cctgagcacc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
      961 gctggagttc gtgaccgccg ccgggatcac tcacggcatg gacgagctgt acaagtaaag
     1021 cggccggccg cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc
     1081 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     1141 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1201 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1261 atcttaaggc gtaaattgta agcgttaatg atccactaga ggatccccgg gtaccgagct
     1321 cgaattcgaa gttcctatac tcctataaga ataggaactt cggaatagga acttcatgat
     1381 ggaagttcat atactttcta gagaatagga acttcggaat aggaacttca taacttcgta
     1441 taggatactt tatacgaagt tatcagctgc cgctcgaggc gcgtgatctt acggcattat
     1501 acgtatgatc ggtccacgat cagctagatt atctagtcag cttgatgtca tagctgtttc
     1561 ctgaggctca atactgacca tttaaatcat acctgacctc catagcagaa agtcaaaagc
     1621 ctccgaccgg aggcttttga cttgatcggc acgtaagagg ttccaacttt caccataatg
     1681 aaataagatc actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag
     1741 ctaaaatgag ccatattcaa cgggaaacgt cttgcttgaa gccgcgatta aattccaaca
     1801 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     1861 caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     1921 gtagcgttgc caatgatgtt acagatgaga tggtcaggct aaactggctg acggaattta
     1981 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     2041 ctgcgatccc agggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     2101 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     2161 gtccttttaa cggcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     2221 gtttggttgg tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     2281 ggaaagaaat gcataaactc ttgccattct caccggattc agtcgtcact catggtgatt
     2341 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     2401 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     2461 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     2521 ataaattgca gtttcacttg atgctcgatg agtttttcta atgaggacct aaatgtaatc
     2581 acctggctca ccttcgggtg ggcctttctg cgttgctggc gtttttccat aggctccgcc
     2641 cccctgacga gcatcacaaa aatcgatgct caagtcagag gtggcgaaac ccgacaggac
     2701 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2761 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2821 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2881 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2941 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3001 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3061 gaagaacagt atttggtatc tgcgctctgc tgaagccagt tacctcggaa aaagagttgg
     3121 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3181 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgattttct accgaagaaa
     3241 ggccca
//
