LOCUS       sequence-381440-        8377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter of cytosolic calcium in C.elegans..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8377)
  TITLE     Subcellular imaging of neuronal calcium handling in vivo
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8377)
  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            "Unpublished"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: pAS1 - pRig-3::GCaMP6f::unc-54
FEATURES             Location/Qualifiers
     source          1..8377
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(2991..2999,3000..3017,3018..3020,3021..3053,3054..3056,
                     3057..3080,3081..3107,3108..3164,3165..3170,3171..3440,
                     3441..3458,3459..3893,3894..3899,3900..4340)
                     /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      3087..3096
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3377..3398
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3504..3525)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3765..3784)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(5298..5316)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5299..5316)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5334..5350)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5334..5350)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5347..5369)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5358..5374
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5382..5388,5389..5406,5407..5412))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5427..5448
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5565..5582)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5736..6324)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5816..5835)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6495..7286,7287..7355))
                     /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     7118..7137
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7356..7460)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7538..7993)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7675..7696)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7887..7906
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8119..8141
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8133..8150
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8134..8150
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8157..8176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8157..8175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      8206..8215
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      8210..8219
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 gtggtcagag acaccacatt gaattaacta ttagttcaaa agttgcaggt ttcctagtgc
       61 aaaaagttta tgttcccatc taccaaacga ctcaccgcca attgactttg cagtggcaac
      121 attgggaaat gcgacccatc atattgttcc tattgtcatg tacaagctgt ccctgttttg
      181 ctcgcttaat ccgtcaaccc cttccccgtt ctaattagag atgagtgaat tacaatagag
      241 ttgaaacgga aatctgagtg tctgtagctt tccgccgccg cttttgttac gagatggtag
      301 acgtcccttt cttggtgttt gttgtctcac aaactgtata ataaagcacc gttgtgtaaa
      361 aggcatttgg tatcaggaag ataaaatgaa taaaatgcaa atgcggaatc aaaaattgcc
      421 aaataatttt gaccaataaa atatcaaaaa taatagaaca gaaataaaca aataaaacta
      481 cggagaaaat gtgattattc cattaaacaa cgttttaaac caactttatt cgtcagtggg
      541 gaaggttttg gacaaatatc atagtaaaac ttgttcgttt taattataca taatcaataa
      601 ttataggtgt tagaaccaat ggtcgaaatt taaaaaaaaa tcaacatttt agaaaatttc
      661 ggcaatttac cgaaatattg actgacaatt ttacatatat aagtgttttg aaatagaatc
      721 cccattgacg atgcgccacc ttaaacagta gaactgttcc aatcagaaac taaataaata
      781 cggttattat atatatatat atataacttg tttttattat gcatttctca aaactcacca
      841 aactcaatct ctaatttacg gttgttgagg aaaatatatt tattacatct agttgagact
      901 ctaaaagaag tacaactttt tcttgcaatt cctccaccaa tccaattttt caagagtcaa
      961 ttgcttggag tcctcgtttt ctcgtaatcc gatattcccc ctatttctgt gattttattc
     1021 agtttcgtcc tcttacacca aaaatcttca tgactttttt ggttttctgc ttaatctttt
     1081 ttttgttttt aaagtctacc atccggcaat tttttttgca aatggttgct cagaatttga
     1141 cctcagatta aaattttcag gaacttcctg gtcccatttc gtcacattta ataacaaacc
     1201 aaatccttca gcttgagtcg tccaagccaa cgcaacccta attcgattcc acctccaatt
     1261 tacctttttg ccgttttcga tcaaattgtg ggggctcttt ttatttgaaa gtcgtaaaat
     1321 ggcgcgtcct tgtcgaaaat tgagatagct aatggttcca aataaaatca aatgcatgaa
     1381 cctagtagga aaaatcgttt tttttttcat cttccaaaga aaaagtttaa gataaaccca
     1441 ccactccagc agtccaccta cgcgtttttc accggaaact gtcatttgtt tttgatgtat
     1501 gacagtgcca gcactgaact aaattcccat ttgttttgtt gtggtgttat ctaaccacgt
     1561 ggctctttct ctaaattttg cgtaggctgc aagatccgca atcccaccaa aaaatggttt
     1621 ctaatgtact tcatacatat cccacatgac tcagtgctcc gaaacgccca gcaacccctg
     1681 cgtttgtctt tacaaaaatg aggcggaaat gggaaatttt cctgctattg ctatggtaaa
     1741 atacttaagc tgtattgaga tttgggaaat tgaaagataa gatgaaaaat ccactctgta
     1801 taattaaaaa ggtttatttt ttttgtaatt tttgtaccgc aaattttttc attaactgat
     1861 ttttctttat tcattcgatg gcccagtttc agatcttcat tatcatgtag tttattcatt
     1921 caaactattt gtgacatcat gatggttgtt taaaaataat ttcgaatgaa gaaagcttaa
     1981 acctattata attttctttt ctataaaaaa tattgatttt gtaaagcgtt tggcagtgac
     2041 agtaccaaaa aatgttttaa aatgatttaa gaaaattttt tattttattt gtataatacg
     2101 cgtaaaatga gtatactcgt catttgtcac gaatcgagtt ctcagtttgc tgtgaatatc
     2161 ttgtaataga aatcgatctc atagtgtcac ttttgaggca taacttttca ggttgttaca
     2221 agttcattaa cttgtcaaga ctgggtcatg ttttaaagtg taactgttac ccatgtaccc
     2281 taaactttga gacaaactcc tcaaaacttg taaccagaat cctatgtaga tgactcacaa
     2341 aaccctgttt tcaatttgaa cgaatttcga aactggccaa aatgttcccc tcttgctcac
     2401 ctaatgcttt taccatttaa aaaggatggt aacaacactc tttcgaaaat gtaggcggtc
     2461 cattttcggt ttctcataac cgtctggctc tatctcctag tgcgcgagga gagggcgggc
     2521 ggagtaggaa tcgggggcag cgcactcata cactctgccc ttctcttttg cacacacaga
     2581 aaaagataga tacagaccaa cacttattgt tgtccagtaa gcttccagaa aactattttc
     2641 agaattacaa gctacatttt tctagaacag tcaattgatt ttttacaaga ttcgagtatt
     2701 ctaacattag tgaaagtatt tagccactgc aattttcaac tgtaggtttc aacttccaaa
     2761 aaaaattcct agcatttcta attggatgaa aaaaggctat ctagaccctc cattatttac
     2821 tcataatttc atctcgtcct catttttaat cccctcattg atcccatcac aatccgccaa
     2881 aatgtttttt aaacactttt ttcttcacca gccgtatgtg gtcaactgcc taaaaatttc
     2941 acagctattt gtcattcctt gcagaagaac ttcattcttc tttttcgaaa atgggttctc
     3001 atcatcatca tcatcatggt atggctagca tgactggtgg acagcaaatg ggtcgggatc
     3061 tgtacgacga tgacgataag gatctcgcca ccatggtcga ctcatcacgt cgtaagtgga
     3121 ataagacagg tcacgcagtc agagctatag gtcggctgag ctcactcgag aacgtctata
     3181 tcaaggccga caagcagaag aacggcatca aggcgaactt caagatccgc cacaacatcg
     3241 aggacggcgg cgtgcagctc gcctaccact accagcagaa cacccccatc ggcgacggcc
     3301 ccgtgctgct gcccgacaac cactacctga gcgtgcagtc caaactttcg aaagacccca
     3361 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     3421 gcatggacga gctgtacaag ggcggtaccg gagggagcat ggtgagcaag ggcgaggagc
     3481 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     3541 tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg caagctgacc ctgaagttca
     3601 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     3661 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     3721 ccatgcccga aggctacatc caggagcgca ccatcttctt caaggacgac ggcaactaca
     3781 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     3841 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aacctgccgg
     3901 accaactgac tgaagagcag atcgcagaat ttaaagagga attctcccta tttgacaagg
     3961 acggggatgg gacaataaca accaaggagc tggggacggt gatgcggtct ctggggcaga
     4021 accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac ggtgacggca
     4081 caatcgactt ccctgagttc ctgacaatga tggcaagaaa aatgaaatac agggacacgg
     4141 aagaagaaat tagagaagcg ttcggtgtgt ttgataagga tggcaatggc tacatcagtg
     4201 cagcagagct tcgccacgtg atgacaaacc ttggagagaa gttaacagat gaagaggttg
     4261 atgaaatgat cagggaagca gacatcgatg gggatggtca ggtaaactac gaagagtttg
     4321 tacaaatgat gacagcgaag tacaagttct gctacgagaa cgaggtgtaa tgacaatcat
     4381 tcgtagaatt ccaactgagc gccggtcgct accattacca acttgtctgg tgtcaaaaat
     4441 aataggggcc gctgtcatca gagtaagttt aaactgagtt ctactaacta acgagtaata
     4501 tttaaatttt cagcatctcg cgcccgtgcc tctgacttct aagtccaatt actcttcaac
     4561 atccctacat gctctttctc cctgtgctcc caccccctat ttttgttatt atcaaaaaac
     4621 ttctcttaat ttctttgttt tttagcttct tttaagtcac ctctaacaat gaaattgtgt
     4681 agattcaaaa atagaattaa ttcgtaataa aaagtcgaaa aaaattgtgc tccctccccc
     4741 cattaataat aattctatcc caaaatctac acaatgttct gtgtacactt cttatgtttt
     4801 ttacttctga taaatttttt tgaaacatca tagaaaaaac cgcacacaaa ataccttatc
     4861 atatgttacg tttcagttta tgaccgcaat ttttatttct tcgcacgtct gggcctctca
     4921 tgacgtcaaa tcatgctcat cgtgaaaaag ttttggagta tttttggaat ttttcaatca
     4981 agtgaaagtt tatgaaatta attttcctgc ttttgctttt tggggtttcc cctattgttt
     5041 gtcaagattt cgaggacggc gtttttcttg ctaaaatcac aagtattgat gagcacgatg
     5101 caagaaagat cggaagaagg tttgggtttg aggctcagtg gaaggtgagt agaagttgat
     5161 aatttgaaag tggagtagtg tctatggggt ttttgcctta aatgacagaa tacattccca
     5221 atataccaaa cataactgtt tcctactagt cggtcgacca tatgggaccc aacgcgttgg
     5281 atgcatagct tgagtattct atagtgtcac ctaaatagct tggcgtaatc atggtcatag
     5341 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5401 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5461 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5521 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5581 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5641 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5701 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5761 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5821 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5881 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5941 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6001 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6061 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6121 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6181 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6241 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6301 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6361 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6421 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6481 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6541 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6601 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6661 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6721 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6781 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6841 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6901 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6961 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7021 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7081 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7141 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7201 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7261 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7321 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7381 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7441 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgatgc ggtgtgaaat
     7501 accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt taatattttg
     7561 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     7621 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     7681 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     7741 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     7801 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     7861 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     7921 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     7981 ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     8041 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     8101 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat
     8161 acgactcact atagggcgaa ttgggcccga cgtcgcatgc tcccggccgc catggcggcc
     8221 gcgggaattc gatttccatg gacaccacgc tcacacacga tgaaattcat acccaccaac
     8281 ctcccttctc tgcctccctc aacttcttcc acggaattct atctcctctt atccagttag
     8341 ggacacatat cgagtttcac caaaaataat gagaagt
//
