LOCUS       sequence-77365-e        7484 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 7484)
  TITLE     Ting GCaMP6 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7484)
  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            "Unpublished"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..7484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(62..650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(142..161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(821..1612,1613..1681))
                     /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     1444..1463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1682..1786)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1854..1872
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1910..1932)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2032..2051
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2068..2523)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2205..2226)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2417..2436
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2598..2738
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2609..2738
                     /label=AAV2 ITR
     polyA_signal    2850..2961
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    2994..3582
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3100..3111
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3509..3529
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(3607..3640)
                     /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(3691..3724)
                     /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      3740..3749
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3746..3754,3755..3772,3773..3775,3776..3808,3809..3811,
                     3812..3835,3836..3862,3863..3919,3920..3925,3926..4195,
                     4196..4213,4214..4648,4649..4654,4655..5095)
                     /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      3842..3851
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     4132..4153
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4259..4280)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4520..4539)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             5111..5167
                     /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             5180..5200
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5201..5899
                     /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(5331..5349)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5599..5618
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5793..5816
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    5917..5950
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    6001..6034
                     /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(6065..7243)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6074..7012
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(6097..6117)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     repeat_region   7344..7484
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7344..7473
                     /label=AAV2 ITR
ORIGIN
        1 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
       61 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      241 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      481 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
      541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      661 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      721 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      781 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      841 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
      901 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
      961 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1021 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1081 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1141 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1201 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1261 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1321 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1381 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1441 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1501 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1561 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1621 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1681 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1741 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1801 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     1861 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     1921 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     1981 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     2041 agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg
     2101 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     2161 cttataaatc aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga
     2221 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     2281 atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag
     2341 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     2401 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     2461 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     2521 cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa
     2581 ataccgcatc aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg
     2641 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
     2701 cagagaggga gtggccaact ccatcactag gggttcctgc ggccgctcgg tccgcacgtg
     2761 catgcctgct attgtcttcc caatcctccc ccttgctgtc ctgccccacc ccacccccca
     2821 gaatagaatg acacctactc agacaatgcg atgcaatttc ctcattttat taggaaagga
     2881 cagtgggagt ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa acaacagatg
     2941 gctggcaact agaaggcaca gtcgagatct ctcgagcagc gctcggtatc gatgcgggga
     3001 ggcggcccaa agggagatcc gactcgtctg agggcgaagg cgaagacgcg gaagaggccg
     3061 cagagccggc agcaggccgc gggaaggaag gtccgctgga ttgagggccg aagggacgta
     3121 gcagaaggac gtcccgcgca gaatccaggt ggcaacatag gcgagcagcc aaggaaagga
     3181 cgatgatttc cccgacaaca ccacggaatt gtcagtgccc aacagccgag cccctgtcca
     3241 gcagcgggca aggcaggcgg cgatgagttc cgccgtggca atagggaggg ggaaagcgaa
     3301 agtcccggaa aggagctgac aggtggtggc aatgccccaa ccagtggggg ttgcgtcagc
     3361 aaacacagtg cacaccacgc cacgttgcct gacaacgggc cacaactcct cataaagaga
     3421 cagcaaccag gatttataca aggaggagaa aatgaaagcc atacgggaag caatagcatg
     3481 atacaaaggc attaaagcag cgtatccaca tagcgtaaaa ggagcaacat agttaagaat
     3541 accagtcaat ctttcacaaa ttttgtaatc cagaggttga ttatcgataa gcttgatatc
     3601 gaattcataa cttcgtatag catacattat acgaagttat tctttgcacc attctaaaga
     3661 ataacagtga taatttctgg gttaaggcaa ataacttcgt ataggatact ttatacgaag
     3721 ttatgctagc agcgctgttg ccaccatggg ttctcatcat catcatcatc atggtatggc
     3781 tagcatgact ggtggacagc aaatgggtcg ggatctgtac gacgatgacg ataaggatct
     3841 cgccaccatg gtcgactcat cacgtcgtaa gtggaataag acaggtcacg cagtcagagc
     3901 tataggtcgg ctgagctcac tcgagaacgt ctatatcaag gccgacaagc agaagaacgg
     3961 catcaaggcg aacttcaaga tccgccacaa catcgaggac ggcggcgtgc agctcgccta
     4021 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     4081 cctgagcgtg cagtccaaac tttcgaaaga ccccaacgag aagcgcgatc acatggtcct
     4141 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggcgg
     4201 taccggaggg agcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     4261 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggtgaggg
     4321 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     4381 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     4441 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acatccagga
     4501 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     4561 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     4621 catcctgggg cacaagctgg agtacaacct gccggaccaa ctgactgaag agcagatcgc
     4681 agaatttaaa gaggaattct ccctatttga caaggacggg gatgggacaa taacaaccaa
     4741 ggagctgggg acggtgatgc ggtctctggg gcagaacccc acagaagcag agctgcagga
     4801 catgatcaat gaagtagatg ccgacggtga cggcacaatc gacttccctg agttcctgac
     4861 aatgatggca agaaaaatga aatacaggga cacggaagaa gaaattagag aagcgttcgg
     4921 tgtgtttgat aaggatggca atggctacat cagtgcagca gagcttcgcc acgtgatgac
     4981 aaaccttgga gagaagttaa cagatgaaga ggttgatgaa atgatcaggg aagcagacat
     5041 cgatggggat ggtcaggtaa actacgaaga gtttgtacaa atgatgacag cgaaggataa
     5101 cggaagcgga gccactaact tctccctgtt gaaacaagca ggggatgtcg aagagaatcc
     5161 cgggccatct agaatggccc ccaagaagaa gaggaaggtg gtgagcaagg gcgaggaggt
     5221 catcaaagag ttcatgcgct tcaaggtgcg catggagggc tccatgaacg gccacgagtt
     5281 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     5341 ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc agttcatgta
     5401 cggctccaag gcgtacgtga agcaccccgc cgacatcccc gattacaaga agctgtcctt
     5461 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggtc tggtgaccgt
     5521 gacccaggac tcctccctgc aggacggcac gctgatctac aaggtgaaga tgcgcggcac
     5581 caacttcccc cccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctccac
     5641 cgagcgcctg tacccccgcg acggcgtgct gaagggcgag atccaccagg ccctgaagct
     5701 gaaggacggc ggccactacc tggtggagtt caagaccatc tacatggcca agaagcccgt
     5761 gcaactgccc ggctactact acgtggacac caagctggac atcacctccc acaacgagga
     5821 ctacaccatc gtggaacagt acgagcgctc cgagggccgc caccacctgt tcctgtacgg
     5881 catggacgag ctgtacaagt aagtcgacgg cgcgccataa cttcgtataa tgtatgctat
     5941 acgaagttat taagaggttt catattgcta atagcagcta caatccagct accattctgc
     6001 ataacttcgt ataaagtatc ctatacgaag ttattccgga gtcgactcta gaggatccgg
     6061 tacctcacga cacctgaaat ggaagaaaaa aactttgaac cactgtctga ggcttgagaa
     6121 tgaaccaaga tccaaactca aaaagggcaa attccaagga gaattacatc aagtgccaag
     6181 ctggcctaac ttcagtctcc acccactcag tgtggggaaa ctccatcgca taaaacccct
     6241 ccccccaacc taaagacgac gtactccaaa agctcgagaa ctaatcgagg tgcctggacg
     6301 gcgcccggta ctccgtggag tcacatgaag cgacggctga ggacggaaag gcccttttcc
     6361 tttgtgtggg tgactcaccc gcccgctctc ccgagcgccg cgtcctccat tttgagctcc
     6421 ctgcagcagg gccgggaagc ggccatcttt ccgctcacgc aactggtgcc gaccgggcca
     6481 gccttgccgc ccagggcggg gcgatacacg gcggcgcgag gccaggcacc agagcaggcc
     6541 ggccagcttg agactacccc cgtccgattc tcggtggccg cgctcgcagg ccccgcctcg
     6601 ccgaacatgt gcgctgggac gcacgggccc cgtcgccgcc cgcggcccca aaaaccgaaa
     6661 taccagtgtg cagatcttgg cccgcattta caagactatc ttgccagaaa aaaagcgtcg
     6721 cagcaggtca tcaaaaattt taaatggcta gagacttatc gaaagcagcg agacaggcgc
     6781 gaaggtgcca ccagattcgc acgcggcggc cccagcgccc aggccaggcc tcaactcaag
     6841 cacgaggcga aggggctcct taagcgcaag gcctcgaact ctcccaccca cttccaaccc
     6901 gaagctcggg atcaagaatc acgtactgca gccaggtgga agtaattcaa ggcacgcaag
     6961 ggccataacc cgtaaagagg ccaggcccgc gggaaccaca cacggcactt acctgtgttc
     7021 tggcggcaaa cccgttgcga aaaagaacgt tcacggcgac tactgcactt atatacggtt
     7081 ctcccccacc ctcgggaaaa aggcggagcc agtacacgac atcactttcc cagtttaccc
     7141 cgcgccacct tctctaggca ccggttcaat tgccgacccc tccccccaac ttctcgggga
     7201 ctgtgggcga tgtgcgctct gcccactgac gggcaccgga gccaattccc actcctttca
     7261 agacctagaa ggtccattag ctgcaaagat tcctctctgt ttaaaacttt atccatcttt
     7321 gcaaagctta cgcgtgcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     7381 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     7441 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg cagg
//
