LOCUS       sequence-189879-        4487 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutively expresses the CaPR construct, the voltage indicator 
            PROPS tethered to the calcium sensor GCaMP6f, under the Sigma 70 
            promoter in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4487)
  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 4487)
  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: pKL001
FEATURES             Location/Qualifiers
     source          1..4487
                     /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"
     primer_bind     complement(1397..1414)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     primer_bind     complement(1611..1628)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1782..2370)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1862..1881)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2541..3332,3333..3401))
                     /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     3164..3183
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3402..3506)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3574..3592
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3596..3639)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    3642..3663
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        3664..3698
                     /label=BBa_J23118
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.56"
     CDS             join(4479..4487,1..18,19..21,22..54,55..57,58..81,82..108,
                     109..165,166..171,172..441,442..459,460..894,895..900,
                     901..1344)
                     /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"
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 atttgacaag
      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 gtaagtttaa acggtctcca gcttggctgt tttggcggat
     1381 gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac
     1441 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     1501 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     1561 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg ccttggcttc ctcgctcact
     1621 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     1681 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     1741 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccacag gctccgcccc
     1801 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1861 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1921 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1981 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     2041 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     2101 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     2161 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     2221 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     2281 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     2341 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     2401 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     2461 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     2521 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     2581 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     2641 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     2701 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     2761 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     2821 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     2881 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     2941 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3001 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     3061 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3121 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3181 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3241 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     3301 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     3361 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     3421 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     3481 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     3541 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gcagaatttc
     3601 agataaaaaa aatccttagc tttcgctaag gatgatttct ggaattcgcg gccgcttcta
     3661 gagttgacgg ctagctcagt cctaggtatt gtgctagcat taactttaag aaggagatat
     3721 acatacccat gggtaaatta ttactgatat taggtagtgt tattgcactt cctacatttg
     3781 ctgcaggtgg tggtgacctt gatgctagtg attacactgg tgtttctttt tggttagtta
     3841 ctgctgcttt attagcatct actgtatttt tctttgttga aagagataga gtttctgcaa
     3901 aatggaaaac atcattaact gtatctggtc ttgttactgg tattgctttc tggcattaca
     3961 tgtacatgag aggggtatgg attgaaactg gtgattcgcc aactgtattt agatacatta
     4021 attggttact aacagttcct ctattaatat gtgaattcta cttaattctt gctgctgcaa
     4081 ctaatgttgc tggatcatta tttaagaaat tactagttgg ttctcttgtt atgcttgtgt
     4141 ttggttacat gggtgaagca ggaatcatgg ctgcatggcc tgcattcatt attgggtgtt
     4201 tagcttgggt atacatgatt tatgaattat gggctggaga aggaaaatct gcatgtaata
     4261 ctgcaagtcc tgctgtgcaa tcagcttaca acacaatgat gtatattatc atctttggtt
     4321 gggcgattta tcctgtaggt tatttcacag gttacctgat gggtgacggt ggatcagctc
     4381 ttaacttaaa ccttatctat aaccttgctg actttgttaa caagattcta tttggtttaa
     4441 ttatatggaa tgttgctgtt aaagaatctt ctaatgctat gggttct
//
