LOCUS       sequence-175189-        7480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre dependent expression of GCaMP6f 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  JOURNAL   Unpublished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            "Unpublished"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-GCaMP6f-P2A-nls-dTomato
FEATURES             Location/Qualifiers
     source          1..7480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    239..350
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    384..972
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             490..501
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     899..919
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(997..1030)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1081..1114)
                     /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)."
     regulatory      1130..1139
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1136..1144,1145..1162,1163..1165,1166..1198,1199..1201,
                     1202..1225,1226..1252,1253..1309,1310..1315,1316..1585,
                     1586..1603,1604..2038,2039..2044,2045..2485)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1232..1241
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1522..1543
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1649..1670)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1910..1929)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2501..2557
                     /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             2570..2590
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2591..3289
                     /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(2721..2739)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2989..3008
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3183..3206
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    3307..3340
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3391..3424
                     /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)."
     promoter        complement(3455..4633)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3464..4402
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3487..3507)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     repeat_region   4734..4863
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4925..5513)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5005..5024)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5684..6475,6476..6544))
                     /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     6307..6326
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6545..6649)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6717..6735
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6773..6795)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6895..6914
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6931..7386)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7068..7089)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7280..7299
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   join(7461..7480,1..121)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(7472..7480,1..121)
                     /label=AAV2 ITR
ORIGIN
        1 cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg
       61 gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc
      121 tgcggccgct cggtccgcac gtgagatctc atgcctgcta ttgtcttccc aatcctcccc
      181 cttgctgtcc tgccccaccc caccccccag aatagaatga cacctactca gacaatgcga
      241 tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca gggtcaagga
      301 aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacag tcgaagatct
      361 ctcgagcagc gctcggtatc gatgcgggga ggcggcccaa agggagatcc gactcgtctg
      421 agggcgaagg cgaagacgcg gaagaggccg cagagccggc agcaggccgc gggaaggaag
      481 gtccgctgga ttgagggccg aagggacgta gcagaaggac gtcccgcgca gaatccaggt
      541 ggcaacatag gcgagcagcc aaggaaagga cgatgatttc cccgacaaca ccacggaatt
      601 gtcagtgccc aacagccgag cccctgtcca gcagcgggca aggcaggcgg cgatgagttc
      661 cgccgtggca atagggaggg ggaaagcgaa agtcccggaa aggagctgac aggtggtggc
      721 aatgccccaa ccagtggggg ttgcgtcagc aaacacagtg cacaccacgc cacgttgcct
      781 gacaacgggc cacaactcct cataaagaga cagcaaccag gatttataca aggaggagaa
      841 aatgaaagcc atacgggaag caatagcatg atacaaaggc attaaagcag cgtatccaca
      901 tagcgtaaaa ggagcaacat agttaagaat accagtcaat ctttcacaaa ttttgtaatc
      961 cagaggttga ttatcgataa gcttgatatc gaattcataa cttcgtatag catacattat
     1021 acgaagttat tctttgcacc attctaaaga ataacagtga taatttctgg gttaaggcaa
     1081 ataacttcgt ataggatact ttatacgaag ttatgctagc agcgctgttg ccaccatggg
     1141 ttctcatcat catcatcatc atggtatggc tagcatgact ggtggacagc aaatgggtcg
     1201 ggatctgtac gacgatgacg ataaggatct cgccaccatg gtcgactcat cacgtcgtaa
     1261 gtggaataag acaggtcacg cagtcagagc tataggtcgg ctgagctcac tcgagaacgt
     1321 ctatatcaag gccgacaagc agaagaacgg catcaaggcg aacttcaaga tccgccacaa
     1381 catcgaggac ggcggcgtgc agctcgccta ccactaccag cagaacaccc ccatcggcga
     1441 cggccccgtg ctgctgcccg acaaccacta cctgagcgtg cagtccaaac tttcgaaaga
     1501 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     1561 tctcggcatg gacgagctgt acaagggcgg taccggaggg agcatggtga gcaagggcga
     1621 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     1681 caagttcagc gtgtccggcg agggtgaggg cgatgccacc tacggcaagc tgaccctgaa
     1741 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     1801 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     1861 gtccgccatg cccgaaggct acatccagga gcgcaccatc ttcttcaagg acgacggcaa
     1921 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     1981 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacct
     2041 gccggaccaa ctgactgaag agcagatcgc agaatttaaa gaggaattct ccctatttga
     2101 caaggacggg gatgggacaa taacaaccaa ggagctgggg acggtgatgc ggtctctggg
     2161 gcagaacccc acagaagcag agctgcagga catgatcaat gaagtagatg ccgacggtga
     2221 cggcacaatc gacttccctg agttcctgac aatgatggca agaaaaatga aatacaggga
     2281 cacggaagaa gaaattagag aagcgttcgg tgtgtttgat aaggatggca atggctacat
     2341 cagtgcagca gagcttcgcc acgtgatgac aaaccttgga gagaagttaa cagatgaaga
     2401 ggttgatgaa atgatcaggg aagcagacat cgatggggat ggtcaggtaa actacgaaga
     2461 gtttgtacaa atgatgacag cgaaggataa cggaagcgga gccactaact tctccctgtt
     2521 gaaacaagca ggggatgtcg aagagaatcc cgggccatct agaatggccc ccaagaagaa
     2581 gaggaaggtg gtgagcaagg gcgaggaggt catcaaagag ttcatgcgct tcaaggtgcg
     2641 catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     2701 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc
     2761 ctgggacatc ctgtcccccc agttcatgta cggctccaag gcgtacgtga agcaccccgc
     2821 cgacatcccc gattacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     2881 gaacttcgag gacggcggtc tggtgaccgt gacccaggac tcctccctgc aggacggcac
     2941 gctgatctac aaggtgaaga tgcgcggcac caacttcccc cccgacggcc ccgtaatgca
     3001 gaagaagacc atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct
     3061 gaagggcgag atccaccagg ccctgaagct gaaggacggc ggccactacc tggtggagtt
     3121 caagaccatc tacatggcca agaagcccgt gcaactgccc ggctactact acgtggacac
     3181 caagctggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgctc
     3241 cgagggccgc caccacctgt tcctgtacgg catggacgag ctgtacaagt aagtcgacgg
     3301 cgcgccataa cttcgtataa tgtatgctat acgaagttat taagaggttt catattgcta
     3361 atagcagcta caatccagct accattctgc ataacttcgt ataaagtatc ctatacgaag
     3421 ttattccgga gtcgactcta gaggatccgg tacctcacga cacctgaaat ggaagaaaaa
     3481 aactttgaac cactgtctga ggcttgagaa tgaaccaaga tccaaactca aaaagggcaa
     3541 attccaagga gaattacatc aagtgccaag ctggcctaac ttcagtctcc acccactcag
     3601 tgtggggaaa ctccatcgca taaaacccct ccccccaacc taaagacgac gtactccaaa
     3661 agctcgagaa ctaatcgagg tgcctggacg gcgcccggta ctccgtggag tcacatgaag
     3721 cgacggctga ggacggaaag gcccttttcc tttgtgtggg tgactcaccc gcccgctctc
     3781 ccgagcgccg cgtcctccat tttgagctcc ctgcagcagg gccgggaagc ggccatcttt
     3841 ccgctcacgc aactggtgcc gaccgggcca gccttgccgc ccagggcggg gcgatacacg
     3901 gcggcgcgag gccaggcacc agagcaggcc ggccagcttg agactacccc cgtccgattc
     3961 tcggtggccg cgctcgcagg ccccgcctcg ccgaacatgt gcgctgggac gcacgggccc
     4021 cgtcgccgcc cgcggcccca aaaaccgaaa taccagtgtg cagatcttgg cccgcattta
     4081 caagactatc ttgccagaaa aaaagcgtcg cagcaggtca tcaaaaattt taaatggcta
     4141 gagacttatc gaaagcagcg agacaggcgc gaaggtgcca ccagattcgc acgcggcggc
     4201 cccagcgccc aggccaggcc tcaactcaag cacgaggcga aggggctcct taagcgcaag
     4261 gcctcgaact ctcccaccca cttccaaccc gaagctcggg atcaagaatc acgtactgca
     4321 gccaggtgga agtaattcaa ggcacgcaag ggccataacc cgtaaagagg ccaggcccgc
     4381 gggaaccaca cacggcactt acctgtgttc tggcggcaaa cccgttgcga aaaagaacgt
     4441 tcacggcgac tactgcactt atatacggtt ctcccccacc ctcgggaaaa aggcggagcc
     4501 agtacacgac atcactttcc cagtttaccc cgcgccacct tctctaggca ccggttcaat
     4561 tgccgacccc tccccccaac ttctcgggga ctgtgggcga tgtgcgctct gcccactgac
     4621 gggcaccgga gccaattccc actcctttca agacctagaa ggtccattag ctgcaaagat
     4681 tcctctctgt ttaaaacttt atccatcttt gcaaagctta cgcgtgcggc cgcaggaacc
     4741 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4801 accaaaggtc gcccgacgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     4861 aggacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4921 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4981 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5041 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5101 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5161 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5221 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5281 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5341 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5401 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5461 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5521 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5581 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5641 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5701 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5761 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5821 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5881 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5941 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6001 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6061 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6121 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6181 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6241 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6301 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6361 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6421 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6481 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6541 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6601 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6661 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     6721 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
     6781 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     6841 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     6901 cagagcagat tgtactgaga gtgcaccata aaattgtaaa cgttaatatt ttgttaaaat
     6961 tcgcgttaaa tttttgttaa atcagctcat tttttaacca atagaccgaa atcggcaaaa
     7021 tcccttataa atcaaaagaa tagcccgaga tagagttgag tgttgttcca gtttggaaca
     7081 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     7141 gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttggggtcg aggtgccgta
     7201 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     7261 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctaag gcgctggcaa
     7321 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     7381 gcgcgtacta tggttgcttt gacgtatgcg gtgtgaaata ccgcacagat gcgtaaggag
     7441 aaaataccgc atcaggcgcc cctgcaggca gctgcgcgct
//
