LOCUS       sequence-396469-        7193 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses eGCaMP+ in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7193)
  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 7193)
  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(plus)
FEATURES             Location/Qualifiers
     source          1..7193
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    25..36
                     /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        40..419
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        421..696
                     /label=chicken beta-actin promoter
     intron          697..1713
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1637..1659
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1721..1740
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1783..1800
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     2082..2103
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2209..2230)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2470..2489)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     intron          3085..3217
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3200..3219
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3226..3814
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3279..3299)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3697..3708)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3841..3858)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3847..3958
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4054..4074)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4054..4072)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4093..4109)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4215..4234)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4280..4335
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4334..4353)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4696..4712)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4720..4736
                     /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(4744..4750,4751..4768,4769..4774))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4789..4810
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4869..5064
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4915..5050
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5070..5204
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5107..5126)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5161..5180
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5272..5289)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5443..6031)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5523..5542)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6202..6993,6994..7062))
                     /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     6825..6844
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7063..7167)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagagtcatc ctcatcgtcg acattgatta ttgactagtt
       61 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
      121 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      181 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      241 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      301 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      361 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      421 tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa
      481 ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg
      541 ggcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      601 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      661 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      721 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      781 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
      841 taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc
      901 cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc
      961 gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
     1021 gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg
     1081 ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt
     1141 gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt tgctgagcac
     1201 ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc
     1261 ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc
     1321 tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc
     1381 agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc
     1441 tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag
     1501 cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc
     1561 cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg
     1621 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
     1681 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg
     1741 ctgtctcatc attttggcaa agaattcggt accgccacca tgcatcatca ccatcatcac
     1801 acgcgtcgca agaagacctt caaggaggtg gccaccgccg tgaagatcat cggccgcctg
     1861 atgggcctga agatcaacgt ctatatcaag gccgacaagc agaagaacgg catcaaggcg
     1921 aacttccaca tccgccacaa catcgaggac ggcggcgtgc agctcgccta ccactaccag
     1981 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcgtg
     2041 gagtccaaac tttcgaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2101 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggcgg taccggaggg
     2161 agcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2221 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggtgaggg cgatgccacc
     2281 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     2341 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     2401 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acatccagga gcgcaccatc
     2461 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2521 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2581 cacaagctgg agtacaacct gccggaccaa ctgactgaag agcagatcgc agaatttaaa
     2641 gagcacttct ccctatttga caaggacggg gatgggacaa taacaaccaa ggagctggtg
     2701 acggtgatgc ggtctctggg gcagaacccc acagaagcag agctgcagga catgatcaat
     2761 gaagtagatg ccgacggtga cggcacaatc gacttccctg agttcctgac aatgatggca
     2821 agaaaaatga aatacaggga cacggaagaa gaaattagag aagcgttcgg tgtgtttgat
     2881 aaggatggca atggctacat cagtgcagca gagcttcgcc acgtgatgac aaaccttgga
     2941 gagaagttaa cagatgaaga ggttgatgaa atgatcaggg aagcagacat cgatggggat
     3001 ggtcaggtaa actacgaaga gtttgtacaa atgatgacag cgaagtaatg tacaagtaaa
     3061 gcggccccgg actcgaggcc gcaggtaagt atcaaggtta caagacaggt ttaaggagac
     3121 caatagaaac tgggcttgtc gagacagaga agactcttgc gtttctgata ggcacctatt
     3181 ggtcttactg acatccactt tgcctttctc tccacaggtg tcgacaatca acctctggat
     3241 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     3301 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     3361 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     3421 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     3481 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     3541 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3601 tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc
     3661 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3721 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     3781 acgagtcgga tctccctttg ggccgcctcc ccgcctggaa ttcgagctcg gtacgatcag
     3841 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3901 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3961 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     4021 aggattggga agacaatagc ccagcttttg ttccctttag tgagggttaa ttgcgcgctt
     4081 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctaa ttcactcctc
     4141 aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat
     4201 accactgaga tcgatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt
     4261 gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat
     4321 tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat
     4381 gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt
     4441 tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat
     4501 agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct
     4561 ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga
     4621 ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct gcagcccaag
     4681 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4741 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4801 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4861 gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     4921 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4981 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     5041 tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca
     5101 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5161 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat
     5221 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5281 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5341 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5401 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5461 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5521 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5581 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5641 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5701 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5761 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5821 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5881 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5941 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6001 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6061 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6121 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6181 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6241 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6301 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6361 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6421 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6481 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6541 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6601 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6661 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6721 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6781 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6841 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6901 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6961 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7021 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7081 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7141 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctg
//
