LOCUS       sequence-311385-        4478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression (sigma 70 promoter) of GCaMP6f tethered to 
            pHuji fluorophores in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4478)
  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 4478)
  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: pKL12-GCaMP6f-pHuji bb118
FEATURES             Location/Qualifiers
     source          1..4478
                     /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(2280..2297)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     primer_bind     complement(2494..2511)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2665..3253)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2745..2764)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3424..4215,4216..4284))
                     /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     4047..4066
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4285..4389)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4457..4475
                     /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 ggtggatccg tttctaaagg tgaagagaac aacatggcta ttatcaaaga
     1561 gttcatgagg tttaaggtcc acatggaagg ttcagtgaac ggccatgagt tcgaaatcga
     1621 gggtgaaggc gaaggtagac catacgaggc gttccagact gccaaactga aagtcactaa
     1681 aggtggacct ttaccgtttg cgtgggatat cctttctcct caatttatgt atggttcaaa
     1741 agtttatatt aaacatccag cagacattcc tgattatttt aaattgtcat tccctgaagg
     1801 ttttagatgg gagagagtca tgaatttcga ggatggtggc ataatccatg ttaatcagga
     1861 ttcttcttta caggatggtg tgttcattta caaagttaag ttaaggggaa ctaactttcc
     1921 ctcagatggt ccagtgatgc aaaagaagac gatgggctgg gaagcttcag aagaaagaat
     1981 gtacccagag gatggcgcct taaagtccga aattaaatat cgtctgaaac tgaaagatgg
     2041 cggccactac gcagctgaag taaagactac ttataaagct aagaaaccag tacagttacc
     2101 cggtgcctat attgtcgata taaaattgga catcgtctct cataatgaag attatactat
     2161 cgttgaacag tacgagaggg ctgagggcag gcactctacc ggtggtatgg acgagttgta
     2221 taaatgagtt taaacggtct ccagcttggc tgttttggcg gatgagagaa gattttcagc
     2281 ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc
     2341 agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc
     2401 gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg
     2461 aaaggctcag tcgaaagact gggccttggc ttcctcgctc actgactcgc tgcgctcggt
     2521 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     2581 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2641 taaaaaggcc gcgttgctgg cgtttttcca caggctccgc ccccctgacg agcatcacaa
     2701 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2761 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2821 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     2881 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     2941 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3001 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3061 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3121 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3181 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3241 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3301 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3361 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3421 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3481 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3541 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     3601 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3661 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3721 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3781 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3841 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3901 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3961 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4021 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4081 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4141 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4201 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4261 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     4321 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4381 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     4441 gacattaacc tataaaaata ggcgtatcac gaggcaga
//
