LOCUS       sequence-189873-        4484 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutively expresses the calcium sensor GCaMP6f tethered to 
            mRuby3 under the Sigma 70 promoter in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4484)
  AUTHORS   Bruni GN, Weekley RA, Dodd BJT, Kralj JM
  TITLE     Voltage-gated calcium flux mediates Escherichia coli 
            mechanosensation.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Aug 29;114(35):9445-9450. doi: 
            10.1073/pnas.1703084114. Epub 2017 Aug 14.
  PUBMED    28808010
REFERENCE   2  (bases 1 to 4484)
  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"; doi: 
            "10.1073/pnas.1703084114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-08-29- 29"; volume: "114"; issue: "35"; pages: 
            "9445-9450"
COMMENT     Sequence Label: pKL004
FEATURES             Location/Qualifiers
     source          1..4484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      88..97
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     378..399
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(505..526)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(766..785)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1378..2088
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYKDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     primer_bind     complement(2144..2161)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     primer_bind     complement(2358..2375)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2529..3117)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2609..2628)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3288..4079,4080..4148))
                     /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     3911..3930
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4149..4253)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4321..4339
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(4343..4386)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    4389..4410
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        4411..4445
                     /label=BBa_J23118
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.56"
     CDS             join(4476..4484,1..18,19..21,22..54,55..57,58..81,82..108,
                     109..165,166..171,172..441,442..459,460..894,895..900,
                     901..1341)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFNKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
ORIGIN
        1 catcatcatc atcatcatgg tatggctagc atgactggtg gacagcaaat gggtcgggat
       61 ctgtacgacg atgacgataa ggatctcgcc accatggtcg actcatcacg tcgtaagtgg
      121 aataagacag gtcacgcagt cagagctata ggtcggctga gctcactcga gaacgtctat
      181 atcaaggccg acaagcagaa gaacggcatc aaggcgaact tcaagatccg ccacaacatc
      241 gaggacggcg gcgtgcagct cgcctaccac taccagcaga acacccccat cggcgacggc
      301 cccgtgctgc tgcccgacaa ccactacctg agcgtgcagt ccaaactttc gaaagacccc
      361 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
      421 ggcatggacg agctgtacaa gggcggtacc ggagggagca tggtgagcaa gggcgaggag
      481 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      541 ttcagcgtgt ccggcgaggg tgagggcgat gccacctacg gcaagctgac cctgaagttc
      601 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
      661 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
      721 gccatgcccg aaggctacat ccaggagcgc accatcttct tcaaggacga cggcaactac
      781 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
      841 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacctgccg
      901 gaccaactga ctgaagagca gatcgcagaa tttaaagagg aattctccct atttaacaag
      961 gacggggatg ggacaataac aaccaaggag ctggggacgg tgatgcggtc tctggggcag
     1021 aaccccacag aagcagagct gcaggacatg atcaatgaag tagatgccga cggtgacggc
     1081 acaatcgact tccctgagtt cctgacaatg atggcaagaa aaatgaaata cagggacacg
     1141 gaagaagaaa ttagagaagc gttcggtgtg tttgataagg atggcaatgg ctacatcagt
     1201 gcagcagagc ttcgccacgt gatgacaaac cttggagaga agttaacaga tgaagaggtt
     1261 gatgaaatga tcagggaagc agacatcgat ggggatggtc aggtaaacta cgaagagttt
     1321 gtacaaatga tgacagcgaa gggtggatcc ggtggtagcg gtggtagcgg tggatccatg
     1381 gtgtctaagg gcgaagagct gatcaaggaa aatatgcgta tgaaggtggt catggaaggt
     1441 tcggtcaacg gccaccaatt caaatgcaca ggtgaaggag aaggcagacc gtacgaggga
     1501 actcaaacca tgaggatcaa agtcatcgag ggaggacccc tgccatttgc ctttgacatt
     1561 cttgccacgt cgttcatgta tggcagccgt acttttatca agtacccggc cgacatccct
     1621 gatttcttta aacagtcctt tcctgagggt tttacttggg aaagagttac gagatacaaa
     1681 gatggtggag tcgtcaccgt cacgcaggac accagccttg aggatggcga gctcgtctac
     1741 aacgtcaagg tcagaggggt aaactttccc tccaatggtc ccgtgatgca gaagaagacc
     1801 aagggttggg agcctaatac agagatgatg tatccagcag atggtggtct gagaggatac
     1861 actgacatcg cactgaaagt tgatggtggt ggccatctgc actgcaactt cgtgacaact
     1921 tacaggtcaa aaaagaccgt cgggaacatc aagatgcccg gtgtccatgc cgttgatcac
     1981 cgcctggaaa ggatcgagga gagtgacaat gaaacctacg tagtgcaacg cgaagtggca
     2041 gttgccaaat acagcaacct tggtggtggc atggacgagc tgtacaagta agtttaaacg
     2101 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat
     2161 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     2221 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     2281 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     2341 gactgggcct tggcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2401 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2461 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2521 ctggcgtttt tccacaggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2581 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2641 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2701 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2761 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2821 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2881 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2941 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3001 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3061 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3121 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3181 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3241 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3301 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3361 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3421 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3481 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3541 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3601 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3661 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3721 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3781 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3841 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3901 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3961 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4021 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4081 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4141 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4201 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4261 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     4321 aataggcgta tcacgaggca gaatttcaga taaaaaaaat ccttagcttt cgctaaggat
     4381 gatttctgga attcgcggcc gcttctagag ttgacggcta gctcagtcct aggtattgtg
     4441 ctagcattaa ctttaagaag gagatataca tacccatggg ttct
//
