LOCUS       sequence-396537-        7277 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses eGCaMP2+ in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7277)
  AUTHORS   Wait SJ, Rappleye M, Lee JD, Goy ME, Smith N, Berndt A
  TITLE     Machine Learning Ensemble Directed Engineering of Genetically 
            Encoded Fluorescent Calcium Indicators.
  JOURNAL   Res Sq. 2023 Aug 7:rs.3.rs-3146778. doi: 
            10.21203/rs.3.rs-3146778/v1. Preprint.
  PUBMED    37609342
REFERENCE   2  (bases 1 to 7277)
  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: "Res Sq. 
            2023 Aug 7:rs.3.rs-3146778. doi: 10.21203/rs.3.rs-3146778/v1. 
            Preprint."
COMMENT     Sequence Label: pCAG_eGCaMP(2+)
FEATURES             Location/Qualifiers
     source          1..7277
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     479..498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(717..821)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    872..883
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     enhancer        887..1266
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1268..1543
                     /label=chicken beta-actin promoter
     intron          1544..2560
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2484..2506
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2568..2587
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2621..2630
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2627..2635,2636..2653,2654..2656,2657..2689,2690..2692,
                     2693..2716,2717..2743,2744..2800,2801..2806,2807..3076,
                     3077..3094,3095..3529,3530..3535,3536..3979)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRVIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDDLTEEQIAEFKEHFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      2723..2732
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3013..3034
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3140..3161)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3401..3420)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     intron          4016..4148
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4131..4150
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    4157..4745
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4210..4230)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4628..4639)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4772..4789)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4778..4889
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4985..5005)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4985..5003)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5024..5040)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5146..5165)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5211..5266
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5265..5284)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5627..5643)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5651..5667
                     /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(5675..5681,5682..5699,5700..5705))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5720..5741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5800..5995
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5846..5981
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6001..6135
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6038..6057)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6092..6111
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6203..6220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6374..6962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6454..6473)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7133..7277,1..647,648..716))
                     /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"
ORIGIN
        1 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
       61 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
      121 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
      181 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
      241 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
      301 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
      361 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
      421 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
      481 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
      541 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
      601 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
      661 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
      721 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
      781 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
      841 ccacctgggt cgacattgat tattgactag agtcatcctc atcgtcgaca ttgattattg
      901 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      961 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     1021 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     1081 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     1141 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     1201 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     1261 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
     1321 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
     1381 gggggggggc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag
     1441 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
     1501 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
     1561 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
     1621 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg
     1681 cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg
     1741 ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg
     1801 gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg
     1861 gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg
     1921 cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc
     1981 agggggtgtg ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc
     2041 tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt
     2101 gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg
     2161 ggagggctcg ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc
     2221 gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc
     2281 ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg
     2341 ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc
     2401 gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct
     2461 tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc
     2521 ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt
     2581 tattgtgctg tctcatcatt ttggcaaaga attcggtacc gccaccatgg gttctcatca
     2641 tcatcatcat catggtatgg ctagcatgac tggtggacag caaatgggtc gggatctgta
     2701 cgacgatgac gataaggatc tcgccaccat ggtcgactca tcacgtcgta agtggaataa
     2761 gacaggtcac gcagtcagag tgataggtcg gctgagctca ctcgagaacg tctatatcaa
     2821 ggccgacaag cagaagaacg gcatcaaggc gaacttccac atccgccaca acatcgagga
     2881 cggcggcgtg cagctcgcct accactacca gcagaacacc cccatcggcg acggccccgt
     2941 gctgctgccc gacaaccact acctgagcgt gcagtccaaa ctttcgaaag accccaacga
     3001 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     3061 ggacgagctg tacaagggcg gtaccggagg gagcatggtg agcaagggcg aggagctgtt
     3121 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     3181 cgtgtccggc gagggtgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     3241 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     3301 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     3361 gcccgaaggc tacatccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     3421 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc ttaagggcat
     3481 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaacc ttcctgacga
     3541 tctgactgaa gagcagatcg cagaatttaa agagcacttc tccctatttg acaaggacgg
     3601 ggatgggaca ataacaacca aggagctggg gacggtgatg cggtctctgg ggcagaaccc
     3661 cacagaagca gagctgcagg acatgatcaa tgaagtagat gccgacggtg acggcacaat
     3721 cgacttccct gagttcctga caatgatggc aagaaaaatg aaatacaggg acacggaaga
     3781 agaaattaga gaagcgttcg gtgtgtttga taaggatggc aatggctaca tcagtgcagc
     3841 agagcttcgc cacgtgatga caaaccttgg agagaagtta acagatgaag aggttgatga
     3901 aatgatcagg gaagcagaca tcgatgggga tggtcaggta aactacgaag agtttgtaca
     3961 aatgatgaca gcgaagtaat gtacaagtaa agcggccccg gactcgaggc cgcaggtaag
     4021 tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt cgagacagag
     4081 aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct
     4141 ctccacaggt gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     4201 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     4261 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     4321 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     4381 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     4441 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     4501 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc
     4561 ctttccatgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     4621 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     4681 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     4741 cccgcctgga attcgagctc ggtacgatca gcctcgactg tgccttctag ttgccagcca
     4801 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4861 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4921 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag cccagctttt
     4981 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     5041 tgtgaaattg ttatccgcta attcactcct caggtgcagg ctgcctatca gaaggtggtg
     5101 gctggtgtgg ccaatgccct ggctcacaaa taccactgag atcgatcttt ttccctctgc
     5161 caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa
     5221 atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata
     5281 tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata
     5341 tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc agtatatgaa
     5401 acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt
     5461 ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt
     5521 tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct
     5581 cttatggaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca tagctgtttc
     5641 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5701 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5761 ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt agtcagcaac
     5821 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     5881 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     5941 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     6001 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     6061 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     6121 tatcatgtct ggatccgctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     6181 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     6241 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     6301 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     6361 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     6421 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     6481 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     6541 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6601 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6661 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6721 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6781 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6841 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6901 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6961 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     7021 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     7081 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     7141 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     7201 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     7261 caatgatacc gcgagac
//
