LOCUS       sequence-311291-        4445 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression (sigma 70 promoter) of GCaMP6f tethered to 
            mScarlet fluorophores in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4445)
  AUTHORS   Bruni GN, Kralj JM
  TITLE     Membrane voltage dysregulation driven by metabolic dysfunction 
            underlies bactericidal activity of aminoglycosides.
  JOURNAL   eLife. 2020 Aug 4;9. pii: 58706. doi: 10.7554/eLife.58706.
  PUBMED    32748785
REFERENCE   2  (bases 1 to 4445)
  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: "eLife."; 
            date: "2020-08-4"; volume: "9. pii: 58706. doi"; pages: " 
            10.7554/eLife.58706"
COMMENT     Sequence Label: pKL09-GCaMP6f-mScarlet bb118
FEATURES             Location/Qualifiers
     source          1..4445
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(1..44)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    47..68
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        69..103
                     /label=BBa_J23118
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.56"
     CDS             join(134..142,143..160,161..163,164..196,197..199,200..223,
                     224..250,251..307,308..313,314..583,584..601,602..1036,
                     1037..1042,1043..1483)
                     /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"
     regulatory      230..239
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     520..541
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(647..668)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(908..927)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2247..2264)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     primer_bind     complement(2461..2478)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2632..3220)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2712..2731)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3391..4182,4183..4251))
                     /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     4014..4033
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4252..4356)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4424..4442
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 atttcagata aaaaaaatcc ttagctttcg ctaaggatga tttctggaat tcgcggccgc
       61 ttctagagtt gacggctagc tcagtcctag gtattgtgct agcattaact ttaagaagga
      121 gatatacata cccatgggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg
      181 tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatctcg ccaccatggt
      241 cgactcatca cgtcgtaagt ggaataagac aggtcacgca gtcagagcta taggtcggct
      301 gagctcactc gagaacgtct atatcaaggc cgacaagcag aagaacggca tcaaggcgaa
      361 cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc actaccagca
      421 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcgtgca
      481 gtccaaactt tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
      541 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggta ccggagggag
      601 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      661 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggtgagggcg atgccaccta
      721 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
      781 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
      841 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac atccaggagc gcaccatctt
      901 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
      961 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1021 caagctggag tacaacctgc cggaccaact gactgaagag cagatcgcag aatttaaaga
     1081 ggaattctcc ctatttaaca aggacgggga tgggacaata acaaccaagg agctggggac
     1141 ggtgatgcgg tctctggggc agaaccccac agaagcagag ctgcaggaca tgatcaatga
     1201 agtagatgcc gacggtgacg gcacaatcga cttccctgag ttcctgacaa tgatggcaag
     1261 aaaaatgaaa tacagggaca cggaagaaga aattagagaa gcgttcggtg tgtttgataa
     1321 ggatggcaat ggctacatca gtgcagcaga gcttcgccac gtgatgacaa accttggaga
     1381 gaagttaaca gatgaagagg ttgatgaaat gatcagggaa gcagacatcg atggggatgg
     1441 tcaggtaaac tacgaagagt ttgtacaaat gatgacagcg aagggtggat ccggtggtag
     1501 cggtggtagc ggtggatcca ttaaagagtt catgcgtttt aaagtacaca tggagggttc
     1561 aatgaacgga cacgaatttg agattgaggg tgagggtgag gggcgtccgt acgaagggac
     1621 acagaccgcc aagttgaagg tcaccaaggg gggacctttg cccttttctt gggacatcct
     1681 ttctccccaa tttatgtacg ggagccgcgc tttcacgaag cacccggccg atatcccgga
     1741 ttattacaaa caatcttttc ccgagggatt taagtgggaa cgtgtgatga atttcgagga
     1801 cggcggagct gtgaccgtga cgcaagacac cagtttggag gatggaacat tgatctacaa
     1861 ggtaaagttg cgtgggacga attttcctcc cgacggtcca gttatgcaaa agaaaactat
     1921 gggctgggaa gccagtacgg agcgcttata tccagaggat ggcgtcctga aaggggatat
     1981 caaaatggca cttcgcctga aggacggcgg gcgttattta gcggacttca agaccacgta
     2041 caaggcaaag aaacctgttc agatgccagg cgcatataac gtagatcgta agttagatat
     2101 cacttcgcat aatgaagatt atacagttgt agaacaatac gagcgcagtg agggtcgcca
     2161 ctctaccggg gggatggacg aattgtacaa gtgagtttaa acggtctcca gcttggctgt
     2221 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt
     2281 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     2341 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta
     2401 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg ccttggcttc
     2461 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2521 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2581 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccacag
     2641 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2701 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2761 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2821 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2881 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2941 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3001 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3061 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3121 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3181 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3241 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3301 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3361 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3421 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3481 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3541 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3601 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3661 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     3721 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     3781 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3841 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3901 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3961 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4021 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4081 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4141 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4201 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4261 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4321 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     4381 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     4441 gcaga
//
