LOCUS       sequence-77364-e        7484 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 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  JOURNAL   Unpublished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            "Unpublished"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-GCaMP6s-P2A-nls-dTomato
FEATURES             Location/Qualifiers
     source          1..7484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1420
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1411
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1368..1388
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1451..1484)
                     /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(1535..1568)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1586..2284)
                     /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(1669..1692)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1867..1886)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2136..2154
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2285..2305)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2318..2374)
                     /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(2390..2830,2831..2836,2837..3271,3272..3289,
                     3290..3559,3560..3565,3566..3622,3623..3649,3650..3673,
                     3674..3676,3677..3709,3710..3712,3713..3730,3731..3739))
                     /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     2946..2965
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3205..3226
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3332..3353)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      3634..3643
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3736..3745
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3761..3794
                     /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    3845..3878
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3903..4491
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3956..3976)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4374..4385)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4524..4635
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4747..4887
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4747..4876
                     /label=AAV2 ITR
     rep_origin      4962..5417
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5049..5068)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5259..5280
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5434..5453)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5553..5575
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5613..5631)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5699..5803
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5804..5872,5873..6664)
                     /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(6022..6041)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6835..7423
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7324..7343
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg
      601 ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg
      661 gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc
      721 tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg
      781 gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc
      841 cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg
      901 agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc
      961 tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc
     1021 agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag
     1081 gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc
     1141 gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga
     1201 ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat
     1261 ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta
     1321 attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac
     1381 agtggttcaa agtttttttc ttccatttca ggtgtcgtga ggtaccggat cctctagagt
     1441 cgactccgga ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg
     1501 attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac
     1561 gaagttatgg cgcgccgtcg acttacttgt acagctcgtc catgccgtac aggaacaggt
     1621 ggtggcggcc ctcggagcgc tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg
     1681 tgatgtccag cttggtgtcc acgtagtagt agccgggcag ttgcacgggc ttcttggcca
     1741 tgtagatggt cttgaactcc accaggtagt ggccgccgtc cttcagcttc agggcctggt
     1801 ggatctcgcc cttcagcacg ccgtcgcggg ggtacaggcg ctcggtggag gcctcccagc
     1861 ccatggtctt cttctgcatt acggggccgt cgggggggaa gttggtgccg cgcatcttca
     1921 ccttgtagat cagcgtgccg tcctgcaggg aggagtcctg ggtcacggtc accagaccgc
     1981 cgtcctcgaa gttcatcacg cgctcccact tgaagccctc ggggaaggac agcttcttgt
     2041 aatcggggat gtcggcgggg tgcttcacgt acgccttgga gccgtacatg aactgggggg
     2101 acaggatgtc ccaggcgaag ggcagggggc cgcccttggt caccttcagc ttggcggtct
     2161 gggtgccctc gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca
     2221 tggagccctc catgcgcacc ttgaagcgca tgaactcttt gatgacctcc tcgcccttgc
     2281 tcaccacctt cctcttcttc ttgggggcca ttctagatgg cccgggattc tcttcgacat
     2341 cccctgcttg tttcaacagg gagaagttag tggctccgct tccgttatcc ttcgctgtca
     2401 tcatttgtac aaactcttcg tagtttacct gaccatcccc atcgatgtct gcttccctga
     2461 tcatttcatc aacctcttca tctgttaact tctctccaag gtttgtcatc acgtggcgaa
     2521 gctctgctgc actgatgtag ccattgccat ccttatcaaa cacaccgaac gcttctctaa
     2581 tttcttcttc cgtgtccctg tatttcattt ttcttgccat cattgtcagg aactcaggga
     2641 agtcgattgt gccgtcaccg tcggcatcta cttcattgat catgtcctgc agctctgctt
     2701 ctgtggggtt ctgccccaga gaccgcatca ccgtccccag ctccttggtt gttattgtcc
     2761 catccccgtc cttgtcaaat agggagaaag cctctttaaa ttctgcgatc tgctcttcag
     2821 tcagttggtc cggcaggttg tactccagct tgtgccccag gatgttgccg tcctccttga
     2881 agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg
     2941 cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg atgtagcctt
     3001 cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc
     3061 actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg
     3121 tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctca ccctcgccgg
     3181 acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc
     3241 cggtgaacag ctcctcgccc ttgctcacca tgctccctcc ggtaccgccc ttgtacagct
     3301 cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc
     3361 gcttctcgtt ggggtctttc gaaagtttgg actgcacgct caggtagtgg ttgtcgggca
     3421 gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtaggcg agctgcacgc
     3481 cgccgtcctc gatgttgtgg cggatgtgga agttcgcctt gatgccgttc ttctgcttgt
     3541 cggccttgat atagacgttc tcgagtgagc tcagccgacc tatagctctg actgcgtgac
     3601 ctgtcttatt ccacttacga cgtgatgagt cgaccatggt ggcgagatcc ttatcgtcat
     3661 cgtcgtacag atcccgaccc atttgctgtc caccagtcat gctagccata ccatgatgat
     3721 gatgatgatg agaacccatg gtggcaacag cgctgctagc ataacttcgt ataaagtatc
     3781 ctatacgaag ttatttgcct taacccagaa attatcactg ttattcttta gaatggtgca
     3841 aagaataact tcgtataatg tatgctatac gaagttatga attcgatatc aagcttatcg
     3901 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     3961 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     4021 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     4081 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     4141 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     4201 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     4261 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     4321 tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     4381 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     4441 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     4501 gagcgctgct cgagagatct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     4561 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4621 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4681 gcaggacagc aagggggagg attgggaaga caatagcagg catgcacgtg cggaccgagc
     4741 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4801 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4861 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     4921 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4981 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     5041 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5101 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5161 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5221 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5281 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc
     5341 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5401 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5461 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5521 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5581 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5641 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5701 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5761 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5821 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5881 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5941 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     6001 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     6061 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     6121 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6181 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6241 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6301 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6361 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6421 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6481 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6541 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6601 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6661 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6721 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6781 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6841 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6901 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6961 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     7021 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     7081 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     7141 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7201 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7261 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7321 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7381 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7441 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
