LOCUS       sequence-176823-        6766 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 6766)
  TITLE     1.	AAV  Synapsin  Intron H2B Gcamp 6f wpre  Pzac2.1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6766)
  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            "Unpublished"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 Gcamp6s reverse
FEATURES             Location/Qualifiers
     source          1..6766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        441..717
                     /label=chicken beta-actin promoter
     primer_bind     996..1018
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1080..1099
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1134..1167
                     /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    1230..1263
                     /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(1272..1715,1716..1721,1722..2156,2157..2174,
                     2175..2444,2445..2450,2451..2507,2508..2534,2535..2558,
                     2559..2561,2562..2594,2595..2597,2598..2615,2616..2624))
                     /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     1831..1850
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2090..2111
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2217..2238)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2519..2528
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2621..2630
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2643..3020)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     protein_bind    complement(3031..3064)
                     /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(3135..3168)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3210..3798
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3263..3283)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3681..3692)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3841..3962
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3852..3871)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3906..3925
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4124..4253
                     /label=AAV2 ITR
     rep_origin      complement(4490..5078)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4570..4589)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5249..6040,6041..6109))
                     /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     5872..5891
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6110..6214)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6240..6695)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6377..6398)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6589..6608
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 aggaagagta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      421 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc
      481 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat
      541 gggggcgggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc
      601 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      661 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      721 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      781 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      841 ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc
      901 cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcggggct gtccgcgggg
      961 ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg
     1021 cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg
     1081 caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc cccataactt
     1141 cgtataaagt atcctatacg aagttatatc aaaataggaa gaccaatgct tcaccatcga
     1201 cccgaattgc caagcatcac catcgaccca taacttcgta taatgtatgc tatacgaagt
     1261 tatactaccg gtcacttcgc tgtcatcatt tgtacaaact cttcgtagtt tacctgacca
     1321 tccccatcga tgtctgcttc cctgatcatt tcatcaacct cttcatctgt taacttctct
     1381 ccaaggtttg tcatcacgtg gcgaagctct gctgcactga tgtagccatt gccatcctta
     1441 tcaaacacac cgaacgcttc tctaatttct tcttccgtgt ccctgtattt catttttctt
     1501 gccatcattg tcaggaactc agggaagtcg attgtgccgt caccgtcggc atctacttca
     1561 ttgatcatgt cctgcagctc tgcttctgtg gggttctgcc ccagagaccg catcaccgtc
     1621 cccagctcct tggttgttat tgtcccatcc ccgtccttgt caaataggga gaaagcctct
     1681 ttaaattctg cgatctgctc ttcagtcagt tggtccggca ggttgtactc cagcttgtgc
     1741 cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg
     1801 gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag
     1861 atggtgcgct cctggatgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc
     1921 atgtggtcgg ggtagcggct gaagcactgc acgccgtagg tcagggtggt cacgagggtg
     1981 ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag
     2041 gtggcatcgc cctcaccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc
     2101 agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct caccatgctc
     2161 cctccggtac cgcccttgta cagctcgtcc atgccgagag tgatcccggc ggcggtcacg
     2221 aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttcgaaag tttggactgc
     2281 acgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg ggtgttctgc
     2341 tggtagtggt aggcgagctg cacgccgccg tcctcgatgt tgtggcggat gtggaagttc
     2401 gccttgatgc cgttcttctg cttgtcggcc ttgatataga cgttctcgag tgagctcagc
     2461 cgacctatag ctctgactgc gtgacctgtc ttattccact tacgacgtga tgagtcgacc
     2521 atggtggcga gatccttatc gtcatcgtcg tacagatccc gacccatttg ctgtccacca
     2581 gtcatgctag ccataccatg atgatgatga tgatgagaac ccatggtggc gactggtgga
     2641 tccttagcgc tggtgtactt ggtgatggcc ttagtaccct cggacacggc gtgcttggcc
     2701 aactccccag gcagcagcag gcgcacggcc gtctggatct ccctggaggt gatggtcgag
     2761 cgcttgttgt aatgcgccag gcgggaagcc tcacctgcga tgcgctcgaa aatgtcgttc
     2821 acaaacgaat tcatgatgcc catggccttg gacgaaatgc cggtgtcagg gtggacctgc
     2881 ttcagaacct tgtacacata gatggaatag ctctccttgc ggctgcgctt gcgcttcttg
     2941 ccgcctttct tctgcgcctt agtcaccgcc ttcttggagc cctttttcgg ggcgggagca
     3001 gacttcgctg gctctggcat accggctagt ataacttcgt ataggatact ttatacgaag
     3061 ttatcattgg gattcttcct attttgatcc aagcatcacc atcgaccctc tagtccagat
     3121 ctcaccatcg acccataact tcgtatagca tacattatac gaagttatgt ccctcgaaga
     3181 ggttcgaatt cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa
     3241 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     3301 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     3361 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     3421 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     3481 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     3541 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     3601 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     3661 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     3721 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     3781 tttgggccgc ctccccgcat cgataccgtc gacccgggcg gccgcttcga gcagacatga
     3841 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     3901 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
     3961 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt
     4021 tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga taaggatctt cctagagcat
     4081 ggctacgtag ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg
     4141 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     4201 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcatt
     4261 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4321 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4381 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4441 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4501 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4561 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4621 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4681 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4741 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4801 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4861 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4921 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4981 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5041 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5101 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5161 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5221 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5281 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5341 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5401 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5461 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5521 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5581 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5641 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5701 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5761 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5821 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5881 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5941 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6001 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6061 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6121 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6181 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     6241 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     6301 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     6361 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     6421 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     6481 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     6541 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     6601 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     6661 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     6721 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccag
//
