LOCUS       sequence-171503-        7979 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid used (after linearization) to generate transgenic mice that 
            express the genetically encoded voltage indicator upon Cre 
            recombination..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7979)
  AUTHORS   Quinn TA, Camelliti P, Rog-Zielinska EA, Siedlecka U, Poggioli T, 
            O'Toole ET, Knopfel T, Kohl P
  TITLE     Electrotonic coupling of excitable and nonexcitable cells in the 
            heart revealed by optogenetics.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Dec 20;113(51):14852-14857. doi: 
            10.1073/pnas.1611184114. Epub 2016 Dec 7.
  PUBMED    27930302
REFERENCE   2  (bases 1 to 7979)
  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"; doi: 
            "10.1073/pnas.1611184114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2016-12-20- 20"; volume: "113"; issue: "51"; pages: 
            "14852-14857"
COMMENT     Sequence Label: pCAG-VSFP2.3 rev. with Flex
FEATURES             Location/Qualifiers
     source          1..7979
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     protein_bind    1789..1822
                     /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)."
     protein_bind    1885..1918
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1934..2644,2645..2647,2648..2650))
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2828..3340,3341..3343,3344..3346))
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIE"
     regulatory      4105..4114
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(4131..4164)
                     /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)."
     protein_bind    complement(4235..4268)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4310..4898
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4363..4383)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4781..4792)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5003..5022)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5068..5123
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5122..5141)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5482..5498)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5482..5498)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5495..5517)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5506..5522
                     /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(5530..5536,5537..5554,5555..5560))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5575..5596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5655..5850
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5701..5836
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5856..5990
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5893..5912)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5947..5966
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6058..6075)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6229..6817)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6309..6328)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6988..7779,7780..7848))
                     /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     7611..7630
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7849..7953)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcgctagc ctgggcaacg
     1741 tgctggttat tgtgctgtct catcattttg gcaaagaatt ggatccccat aacttcgtat
     1801 aaagtatcct atacgaagtt atatcaaaat aggaagacca atgcttcacc atcgacccga
     1861 attgccaagc atcaccatcg acccataact tcgtataatg tatgctatac gaagttatac
     1921 taggatcccc ttacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga
     1981 actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggt
     2041 agctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct
     2101 ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca
     2161 ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt
     2221 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
     2281 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
     2341 cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
     2401 agtcgtgctg cttcatgtgg tcggggtagc gcgcgaagca catcaggccg tagccgaagg
     2461 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg
     2521 tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt
     2581 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
     2641 tgctcaccat gagagtgatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc
     2701 gcttctcgtt ggggtctttg ctcagcttgg actgggtgct caggtagtgg ttgtcgggca
     2761 gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcg agctgcacgc
     2821 tgccgtcctc gatgttgtgg cggatcttga agttggcctt gatgccgttc ttctgcttgt
     2881 cggcggtgat atagacgttg tcgctgatgg cgttgtactc cagcttgtgc cccaggatgt
     2941 tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg gtgtcgccct
     3001 cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct
     3061 cctggacgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc atgtggtcgg
     3121 ggtagcgggc gaagcactgc acgccccagg tcagggtggt cacgagggtg ggccagggca
     3181 cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag gtggcatcgc
     3241 cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca
     3301 ggatgggcac caccccggtg aacagctcct cgcccttgct caccattcga cgcttgttct
     3361 gtgatattgt tcttctgctt gaagccttca tttgttggtg ggaataaaat attcttgcta
     3421 atcgaaccac acgcagcaaa cgggccaaca caaccaactg ccccaaacca tcggctcctg
     3481 tttcttgcac gtattcatct aacacagtgt aaaatatcgt gacgacgaat gtgacaacga
     3541 taatcaaacc atcagcaacc tcccaggggt tggtgaagaa attcttgggc ccgtaggcaa
     3601 atatccttaa tcctaaatcc agcatgaaat aacaagaaag agccaaagcc atcccgtcat
     3661 aaaaggattg tgaagattca ctttttcctg gaagactgag atcaatgatc atgaggatga
     3721 tgtccaagaa aattaggaag actccaaaga ctcgcatccc tagatgatca atcactgctc
     3781 ggacacgaaa ctggacgcga cctacaccag tagtaggtat ctccattctc ccatcgtcca
     3841 ctccatgttc attctcgccc caccatagac ctgaaatctc gggtatatct attcgttcct
     3901 cctgtcttgt ctctggtttt tcactcggtc cttggtctac atcattccaa tgaactttct
     3961 ttaccgattc actttttctt ggcgcagctt tcggcggagc agtgacgtca ccatttatcg
     4021 tgacgtcaac gacgttccgc atgactgcac cgtcaacgcc aactaaatca gctggaggac
     4081 taaaatctga accgtcgaat ccctccatgg tggcggtacc gaattctagt ataacttcgt
     4141 ataggatact ttatacgaag ttatcattgg gattcttcct attttgatcc aagcatcacc
     4201 atcgaccctc tagtccagat ctcaccatcg acccataact tcgtatagca tacattatac
     4261 gaagttatgt ccctcgaaga ggttcgaatt cgatatcaag cttatcgata atcaacctct
     4321 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     4381 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     4441 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     4501 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     4561 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     4621 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     4681 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     4741 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     4801 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     4861 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gcttaaggcg
     4921 gccgcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     4981 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     5041 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     5101 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     5161 atcagaatga gtatttggtt tagagtttgg caacatatgc catatgctgg ctgccatgaa
     5221 caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     5281 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     5341 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     5401 tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct cgacctgcag
     5461 cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5521 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     5581 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5641 gtgccagcgg atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     5701 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     5761 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     5821 ggcttttttg gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg
     5881 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5941 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta
     6001 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     6061 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     6121 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     6181 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6241 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6301 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     6361 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     6421 tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     6481 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6541 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6601 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6661 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6721 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6781 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6841 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6901 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6961 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     7021 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     7081 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     7141 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7201 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7261 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7321 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     7381 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7441 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7501 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7561 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7621 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7681 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7741 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7801 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7861 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7921 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctg
//
