LOCUS       sequence-388695-        8378 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 8378)
  TITLE     Subcellular imaging of neuronal calcium handling in vivo
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8378)
  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            "Unpublished"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..8378
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(3007..3015,3016..3033,3034..3036,3037..3069,3070..3072,
                     3073..3096,3097..3123,3124..3180,3181..3186,3187..3456,
                     3457..3474,3475..3909,3910..3915,3916..4356)
                     /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      3103..3112
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3393..3414
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3520..3541)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3781..3800)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(5314..5332)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5315..5332)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5350..5366)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5350..5366)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5363..5385)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5374..5390
                     /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(5398..5404,5405..5422,5423..5428))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5443..5464
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5581..5598)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5752..6340)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5832..5851)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6511..7302,7303..7371))
                     /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     7134..7153
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7372..7476)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7554..8009)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7691..7712)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7903..7922
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8135..8157
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8149..8166
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8150..8166
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8173..8192
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8173..8191
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      8222..8231
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      8226..8235
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 aaaataatga gaagtgtggt cagagacacc acattgaatt aactattagt tcaaaagttg
       61 caggtttcct agtgcaaaaa gtttatgttc ccatctacca aacgactcac cgccaattga
      121 ctttgcagtg gcaacattgg gaaatgcgac ccatcatatt gttcctattg tcatgtacaa
      181 gctgtccctg ttttgctcgc ttaatccgtc aaccccttcc ccgttctaat tagagatgag
      241 tgaattacaa tagagttgaa acggaaatct gagtgtctgt agctttccgc cgccgctttt
      301 gttacgagat ggtagacgtc cctttcttgg tgtttgttgt ctcacaaact gtataataaa
      361 gcaccgttgt gtaaaaggca tttggtatca ggaagataaa atgaataaaa tgcaaatgcg
      421 gaatcaaaaa ttgccaaata attttgacca ataaaatatc aaaaataata gaacagaaat
      481 aaacaaataa aactacggag aaaatgtgat tattccatta aacaacgttt taaaccaact
      541 ttattcgtca gtggggaagg ttttggacaa atatcatagt aaaacttgtt cgttttaatt
      601 atacataatc aataattata ggtgttagaa ccaatggtcg aaatttaaaa aaaaatcaac
      661 attttagaaa atttcggcaa tttaccgaaa tattgactga caattttaca tatataagtg
      721 ttttgaaata gaatccccat tgacgatgcg ccaccttaaa cagtagaact gttccaatca
      781 gaaactaaat aaatacggtt attatatata tatatatata acttgttttt attatgcatt
      841 tctcaaaact caccaaactc aatctctaat ttacggttgt tgaggaaaat atatttatta
      901 catctagttg agactctaaa agaagtacaa cttttttctt gcaattcctc caccaatcca
      961 atttttcaag agtcaattgc ttggagtcct cgttttctcg taatccgata ttccccctat
     1021 ttctgtgatt ttattcagtt tcgtcctctt acaccaaaaa tcttcatgac ttttttggtt
     1081 ttctgcttaa tctttttttt gtttttaaag tctaccatcc ggcaattttt tttgcaaatg
     1141 gttgctcaga atttgacctc agattaaaat tttcaggaac ttcctggtcc catttcgtca
     1201 catttaataa caaaccaaat ccttcagctt gagtcgtcca agccaacgca accctaattc
     1261 gattccacct ccaatttacc tttttgccgt tttcgatcaa attgtggggg ctctttttat
     1321 ttgaaagtcg taaaatggcg cgtccttgtc gaaaattgag atagctaatg gttccaaata
     1381 aaatcaaatg catgaaccta gtaggaaaaa tcgttttttt tttcatcttc caaagaaaaa
     1441 gtttaagata aacccaccac tccagcagtc cacctacgcg tttttcaccg gaaactgtca
     1501 tttgtttttg atgtatgaca gtgccagcac tgaactaaat tcccatttgt tttgttgtgg
     1561 tgttatctaa ccacgtggct ctttctctaa attttgcgta ggctgcaaga tccgcaatcc
     1621 caccaaaaaa tggtttctaa tgtacttcat acatatccca catgactcag tgctccgaaa
     1681 cgcccagcaa cccctgcgtt tgtctttaca aaaatgaggc ggaaatggga aattttcctg
     1741 ctattgctat ggtaaaatac ttaagctgta ttgagatttg ggaaattgaa agataagatg
     1801 aaaaatccac tctgtataat taaaaaggtt tatttttttt gtaatttttg taccgcaaat
     1861 tttttcatta actgattttt ctttattcat tcgatggccc agtttcagat cttcattatc
     1921 atgtagttta ttcattcaaa ctatttgtga catcatgatg gttgtttaaa aataatttcg
     1981 aatgaagaaa gcttaaacct attataattt tcttttctat aaaaaatatt gattttgtaa
     2041 agcgtttggc agtgacagta ccaaaaaatg ttttaaaatg atttaagaaa attttttatt
     2101 ttatttgtat aatacgcgta aaatgagtat actcgtcatt tgtcacgaat cgagttctca
     2161 gtttgctgtg aatatcttgt aatagaaatc gatctcatag tgtcactttt gaggcataac
     2221 ttttcaggtt gttacaagtt cattaacttg tcaagactgg gtcatgtttt aaagtgtaac
     2281 tgttacccat gtaccctaaa ctttgagaca aactcctcaa aacttgtaac cagaatccta
     2341 tgtagatgac tcacaaaacc ctgttttcaa tttgaacgaa tttcgaaact ggccaaaatg
     2401 ttcccctctt gctcacctaa tgcttttacc atttaaaaag gatggtaaca acactctttc
     2461 gaaaatgtag gcggtccatt ttcggtttct cataaccgtc tggctctatc tcctagtgcg
     2521 cgaggagagg gcgggcggag taggaatcgg gggcagcgca ctcatacact ctgcccttct
     2581 cttttgcaca cacagaaaaa gatagataca gaccaacact tattgttgtc cagtaagctt
     2641 ccagaaaact attttcagaa ttacaagcta catttttcta gaacagtcaa ttgatttttt
     2701 acaagattcg agtattctaa cattagtgaa agtatttagc cactgcaatt ttcaactgta
     2761 ggtttcaact tccaaaaaaa attcctagca tttctaattg gatgaaaaaa ggctatctag
     2821 accctccatt atttactcat aatttcatct cgtcctcatt tttaatcccc tcattgatcc
     2881 catcacaatc cgccaaaatg ttttttaaac acttttttct tcaccagccg tatgtggtca
     2941 actgcctaaa aatttcacag ctatttgtca ttccttgcag aagaacttca ttcttctttt
     3001 tcgaaaatgg gttctcatca tcatcatcat catggtatgg ctagcatgac tggtggacag
     3061 caaatgggtc gggatctgta cgacgatgac gataaggatc tcgccaccat ggtcgactca
     3121 tcacgtcgta agtggaataa gacaggtcac gcagtcagag ctataggtcg gctgagctca
     3181 ctcgagaacg tctatatcaa ggccgacaag cagaagaacg gcatcaaggc gaacttcaag
     3241 atccgccaca acatcgagga cggcggcgtg cagctcgcct accactacca gcagaacacc
     3301 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcgt gcagtccaaa
     3361 ctttcgaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     3421 gccgggatca ctctcggcat ggacgagctg tacaagggcg gtaccggagg gagcatggtg
     3481 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     3541 gtaaacggcc acaagttcag cgtgtccggc gagggtgagg gcgatgccac ctacggcaag
     3601 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     3661 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     3721 gacttcttca agtccgccat gcccgaaggc tacatccagg agcgcaccat cttcttcaag
     3781 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     3841 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     3901 gagtacaacc tgccggacca actgactgaa gagcagatcg cagaatttaa agaggaattc
     3961 tccctatttg acaaggacgg ggatgggaca ataacaacca aggagctggg gacggtgatg
     4021 cggtctctgg ggcagaaccc cacagaagca gagctgcagg acatgatcaa tgaagtagat
     4081 gccgacggtg acggcacaat cgacttccct gagttcctga caatgatggc aagaaaaatg
     4141 aaatacaggg acacggaaga agaaattaga gaagcgttcg gtgtgtttga taaggatggc
     4201 aatggctaca tcagtgcagc agagcttcgc cacgtgatga caaaccttgg agagaagtta
     4261 acagatgaag aggttgatga aatgatcagg gaagcagaca tcgatgggga tggtcaggta
     4321 aactacgaag agtttgtaca aatgatgaca gcgaagtaca agttctgcta cgagaacgag
     4381 gtgtaatgac aatcattcgt agaattccaa ctgagcgccg gtcgctacca ttaccaactt
     4441 gtctggtgtc aaaaataata ggggccgctg tcatcagagt aagtttaaac tgagttctac
     4501 taactaacga gtaatattta aattttcagc atctcgcgcc cgtgcctctg acttctaagt
     4561 ccaattactc ttcaacatcc ctacatgctc tttctccctg tgctcccacc ccctattttt
     4621 gttattatca aaaaacttct cttaatttct ttgtttttta gcttctttta agtcacctct
     4681 aacaatgaaa ttgtgtagat tcaaaaatag aattaattcg taataaaaag tcgaaaaaaa
     4741 ttgtgctccc tccccccatt aataataatt ctatcccaaa atctacacaa tgttctgtgt
     4801 acacttctta tgttttttac ttctgataaa tttttttgaa acatcataga aaaaaccgca
     4861 cacaaaatac cttatcatat gttacgtttc agtttatgac cgcaattttt atttcttcgc
     4921 acgtctgggc ctctcatgac gtcaaatcat gctcatcgtg aaaaagtttt ggagtatttt
     4981 tggaattttt caatcaagtg aaagtttatg aaattaattt tcctgctttt gctttttggg
     5041 gtttccccta ttgtttgtca agatttcgag gacggcgttt ttcttgctaa aatcacaagt
     5101 attgatgagc acgatgcaag aaagatcgga agaaggtttg ggtttgaggc tcagtggaag
     5161 gtgagtagaa gttgataatt tgaaagtgga gtagtgtcta tggggttttt gccttaaatg
     5221 acagaataca ttcccaatat accaaacata actgtttcct actagtcggt cgaccatatg
     5281 ggacccaacg cgttggatgc atagcttgag tattctatag tgtcacctaa atagcttggc
     5341 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5401 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5461 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5521 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5581 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5641 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5701 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5761 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5821 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5881 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5941 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     6001 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6061 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6121 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6181 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6241 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6301 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6361 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6421 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6481 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6541 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6601 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6661 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6721 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6781 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6841 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6901 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6961 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     7021 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     7081 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     7141 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     7201 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     7261 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7321 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7381 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7441 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7501 tgatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt
     7561 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     7621 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     7681 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     7741 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     7801 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     7861 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     7921 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     7981 cgcgcttaat gcgccgctac agggcgcgtc cattcgccat tcaggctgcg caactgttgg
     8041 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     8101 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     8161 gccagtgaat tgtaatacga ctcactatag ggcgaattgg gcccgacgtc gcatgctccc
     8221 ggccgccatg gcggccgcgg gaattcgatt tccatggaca ccacgctcac acacgatgaa
     8281 attcataccc accaacctcc cttctctgcc tccctcaact tcttccacgg aattctatct
     8341 cctcttatcc agttagggac acatatcgag tttcacca
//
