LOCUS       sequence-175187-        7480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre dependent expression of GCaMP6s Ca sensor and physically 
            separate nuclear localized dTomato fluorophore.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7480)
  TITLE     Ting GCaMP6 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7480)
  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: 
            "Unpublished"
COMMENT     Sequence Label: AAV-EF1a-DIO-GCaMP6s-P2A-nls-dTomato
FEATURES             Location/Qualifiers
     source          1..7480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    95..206
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   324..464
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   324..453
                     /label=AAV2 ITR
     rep_origin      539..994
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(626..645)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     836..857
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1011..1030)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1130..1152
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1190..1208)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1276..1380
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1381..1449,1450..2241)
                     /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     complement(1599..1618)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2412..3000
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2901..2920
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   3062..3191
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        3292..4470
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3523..4461
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4418..4438
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(4501..4534)
                     /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(4585..4618)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(4636..5334)
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL
                     VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
                     ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
                     LFLYGMDELYK"
     primer_bind     complement(4719..4742)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(4917..4936)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5186..5204
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(5335..5355)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5368..5424)
                     /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             complement(join(5440..5880,5881..5886,5887..6321,6322..6339,
                     6340..6609,6610..6615,6616..6672,6673..6699,6700..6723,
                     6724..6726,6727..6759,6760..6762,6763..6780,6781..6789))
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     5996..6015
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6255..6276
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6382..6403)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      6684..6693
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      6786..6795
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    6811..6844
                     /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    6895..6928
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    join(6953..7480,1..61)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7006..7026)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7424..7435)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
       61 catcgatacc gagcgctgct cgagagatct tcgactgtgc cttctagttg ccagccatct
      121 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
      181 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
      241 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgagatc
      301 tcacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
      361 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
      421 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
      481 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
      541 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
      601 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
      661 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
      721 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
      781 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
      841 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa
      901 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac
      961 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     1021 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     1081 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     1141 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     1201 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     1261 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1321 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     1381 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     1441 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     1501 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     1561 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     1621 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     1681 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     1741 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     1801 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     1861 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     1921 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     1981 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     2041 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     2101 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     2161 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     2221 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     2281 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     2341 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2401 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2461 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2521 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     2581 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     2641 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     2701 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     2761 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     2821 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     2881 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     2941 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3001 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3061 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt
     3121 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
     3181 taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt ttaaacagag
     3241 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt
     3301 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     3361 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     3421 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     3481 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
     3541 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
     3601 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
     3661 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
     3721 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
     3781 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
     3841 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
     3901 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
     3961 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
     4021 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     4081 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     4141 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     4201 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     4261 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     4321 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     4381 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     4441 agtttttttc ttccatttca ggtgtcgtga ggtaccggat cctctagagt cgactccgga
     4501 ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg attgtagctg
     4561 ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac gaagttatgg
     4621 cgcgccgtcg acttacttgt acagctcgtc catgccgtac aggaacaggt ggtggcggcc
     4681 ctcggagcgc tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg tgatgtccag
     4741 cttggtgtcc acgtagtagt agccgggcag ttgcacgggc ttcttggcca tgtagatggt
     4801 cttgaactcc accaggtagt ggccgccgtc cttcagcttc agggcctggt ggatctcgcc
     4861 cttcagcacg ccgtcgcggg ggtacaggcg ctcggtggag gcctcccagc ccatggtctt
     4921 cttctgcatt acggggccgt cgggggggaa gttggtgccg cgcatcttca ccttgtagat
     4981 cagcgtgccg tcctgcaggg aggagtcctg ggtcacggtc accagaccgc cgtcctcgaa
     5041 gttcatcacg cgctcccact tgaagccctc ggggaaggac agcttcttgt aatcggggat
     5101 gtcggcgggg tgcttcacgt acgccttgga gccgtacatg aactgggggg acaggatgtc
     5161 ccaggcgaag ggcagggggc cgcccttggt caccttcagc ttggcggtct gggtgccctc
     5221 gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca tggagccctc
     5281 catgcgcacc ttgaagcgca tgaactcttt gatgacctcc tcgcccttgc tcaccacctt
     5341 cctcttcttc ttgggggcca ttctagatgg cccgggattc tcttcgacat cccctgcttg
     5401 tttcaacagg gagaagttag tggctccgct tccgttatcc ttcgctgtca tcatttgtac
     5461 aaactcttcg tagtttacct gaccatcccc atcgatgtct gcttccctga tcatttcatc
     5521 aacctcttca tctgttaact tctctccaag gtttgtcatc acgtggcgaa gctctgctgc
     5581 actgatgtag ccattgccat ccttatcaaa cacaccgaac gcttctctaa tttcttcttc
     5641 cgtgtccctg tatttcattt ttcttgccat cattgtcagg aactcaggga agtcgattgt
     5701 gccgtcaccg tcggcatcta cttcattgat catgtcctgc agctctgctt ctgtggggtt
     5761 ctgccccaga gaccgcatca ccgtccccag ctccttggtt gttattgtcc catccccgtc
     5821 cttgtcaaat agggagaaag cctctttaaa ttctgcgatc tgctcttcag tcagttggtc
     5881 cggcaggttg tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc
     5941 cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt
     6001 gtagttgccg tcgtccttga agaagatggt gcgctcctgg atgtagcctt cgggcatggc
     6061 ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc
     6121 gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat
     6181 gaacttcagg gtcagcttgc cgtaggtggc atcgccctca ccctcgccgg acacgctgaa
     6241 cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag
     6301 ctcctcgccc ttgctcacca tgctccctcc ggtaccgccc ttgtacagct cgtccatgcc
     6361 gagagtgatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt
     6421 ggggtctttc gaaagtttgg actgcacgct caggtagtgg ttgtcgggca gcagcacggg
     6481 gccgtcgccg atgggggtgt tctgctggta gtggtaggcg agctgcacgc cgccgtcctc
     6541 gatgttgtgg cggatgtgga agttcgcctt gatgccgttc ttctgcttgt cggccttgat
     6601 atagacgttc tcgagtgagc tcagccgacc tatagctctg actgcgtgac ctgtcttatt
     6661 ccacttacga cgtgatgagt cgaccatggt ggcgagatcc ttatcgtcat cgtcgtacag
     6721 atcccgaccc atttgctgtc caccagtcat gctagccata ccatgatgat gatgatgatg
     6781 agaacccatg gtggcaacag cgctgctagc ataacttcgt ataaagtatc ctatacgaag
     6841 ttatttgcct taacccagaa attatcactg ttattcttta gaatggtgca aagaataact
     6901 tcgtataatg tatgctatac gaagttatga attcgatatc aagcttatcg ataatcaacc
     6961 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     7021 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     7081 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     7141 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     7201 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     7261 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     7321 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt
     7381 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     7441 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
//
