LOCUS       sequence-139154-        6771 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Calcium Sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6771)
  TITLE     1.	AAV  Synapsin  Intron H2B Gcamp 6f wpre  Pzac2.1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6771)
  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            "Unpublished"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 Cag Flex H2B Gcamp6f  reverse
FEATURES             Location/Qualifiers
     source          1..6771
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1932..2387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2069..2090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2281..2300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2459..2588
                     /label=AAV2 ITR
     promoter        2901..3178
                     /label=chicken beta-actin promoter
     primer_bind     3458..3480
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3542..3561
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    3596..3629
                     /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    3692..3725
                     /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(3735..4178,4179..4184,4185..4619,4620..4637,
                     4638..4907,4908..4913,4914..4970,4971..4997,4998..5021,
                     5022..5024,5025..5057,5058..5060,5061..5078,5079..5087))
                     /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"
     primer_bind     4294..4313
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4553..4574
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4680..4701)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      4982..4991
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      5084..5093
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(5106..5483)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     protein_bind    complement(5494..5527)
                     /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(5598..5631)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5673..6261
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5726..5746)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6144..6155)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6304..6425
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6315..6334)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6369..6388
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   6587..6716
                     /label=AAV2 ITR
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg
     2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag
     2641 gaagatcgta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     2701 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     2761 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     2821 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     2881 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc
     2941 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat
     3001 gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg
     3061 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc
     3121 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg
     3181 agtcgctgcg acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
     3241 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
     3301 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
     3361 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ctgtccgcgg
     3421 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     3481 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     3541 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgga tccccataac
     3601 ttcgtataaa gtatcctata cgaagttata tcaaaatagg aagaccaatg cttcaccatc
     3661 gacccgaatt gccaagcatc accatcgacc cataacttcg tataatgtat gctatacgaa
     3721 gttatactag ccggtcactt cgctgtcatc atttgtacaa actcttcgta gtttacctga
     3781 ccatccccat cgatgtctgc ttccctgatc atttcatcaa cctcttcatc tgttaacttc
     3841 tctccaaggt ttgtcatcac gtggcgaagc tctgctgcac tgatgtagcc attgccatcc
     3901 ttatcaaaca caccgaacgc ttctctaatt tcttcttccg tgtccctgta tttcattttt
     3961 cttgccatca ttgtcaggaa ctcagggaag tcgattgtgc cgtcaccgtc ggcatctact
     4021 tcattgatca tgtcctgcag ctctgcttct gtggggttct gccccagaga ccgcatcacc
     4081 gtccccagct ccttggttgt tattgtccca tccccgtcct tgtcaaatag ggagaattcc
     4141 tctttaaatt ctgcgatctg ctcttcagtc agttggtccg gcaggttgta ctccagcttg
     4201 tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc
     4261 agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag
     4321 aagatggtgc gctcctggat gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc
     4381 ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg
     4441 gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg
     4501 taggtggcat cgccctcacc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg
     4561 tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg
     4621 ctccctccgg taccgccctt gtacagctcg tccatgccga gagtgatccc ggcggcggtc
     4681 acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttcga aagtttggac
     4741 tgcacgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc
     4801 tgctggtagt ggtaggcgag ctgcacgccg ccgtcctcga tgttgtggcg gatcttgaag
     4861 ttcgccttga tgccgttctt ctgcttgtcg gccttgatat agacgttctc gagtgagctc
     4921 agccgaccta tagctctgac tgcgtgacct gtcttattcc acttacgacg tgatgagtcg
     4981 accatggtgg cgagatcctt atcgtcatcg tcgtacagat cccgacccat ttgctgtcca
     5041 ccagtcatgc tagccatacc atgatgatga tgatgatgag aacccatggt ggcgactggt
     5101 ggatccttag cgctggtgta cttggtgatg gccttagtac cctcggacac ggcgtgcttg
     5161 gccaactccc caggcagcag caggcgcacg gccgtctgga tctccctgga ggtgatggtc
     5221 gagcgcttgt tgtaatgcgc caggcgggaa gcctcacctg cgatgcgctc gaaaatgtcg
     5281 ttcacaaacg aattcatgat gcccatggcc ttggacgaaa tgccggtgtc agggtggacc
     5341 tgcttcagaa ccttgtacac atagatggaa tagctctcct tgcggctgcg cttgcgcttc
     5401 ttgccgcctt tcttctgcgc cttagtcacc gccttcttgg agcccttttt cggggcggga
     5461 gcagacttcg ctggctctgg cataccggct agtataactt cgtataggat actttatacg
     5521 aagttatcat tgggattctt cctattttga tccaagcatc accatcgacc ctctagtcca
     5581 gatctcacca tcgacccata acttcgtata gcatacatta tacgaagtta tgtccctcga
     5641 agaggttcga attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg
     5701 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     5761 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     5821 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     5881 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     5941 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     6001 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     6061 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     6121 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     6181 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     6241 ccctttgggc cgcctccccg catcgatacc gtcgacccgg gcggccgctt cgagcagaca
     6301 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     6361 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     6421 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg
     6481 ttttttaaag caagtaaaac ctctacaaat gtggtaaaat cgataaggat cttcctagag
     6541 catggctacg tagataagta gcatggcggg ttaatcatta actacaagga acccctagtg
     6601 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     6661 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     6721 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc t
//
