LOCUS       sequence-189876-        7450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET DUET backbone with PROPS voltage indicator and GCaMP6f calcium 
            sensor in the two MCS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7450)
  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 7450)
  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: pKL002
FEATURES             Location/Qualifiers
     source          1..7450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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)."
     RBS             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             81..98
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        964..982
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    983..1007
                     /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)."
     CDS             1056..1088
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(1092..1100,1101..1115)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      1122..1131
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1143..1199
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     1412..1433
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1539..1560)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1800..1819)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2397..2441
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2479..2497)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2493..2540
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2577..3032
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2664..2683)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2874..2895
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3147..3938,3939..4007))
                     /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     3770..3789
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4008..4100)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4181..4769
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4670..4689
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4923..4940
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4955..5094
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5020..5039)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5180..5202
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5196..5387)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5959..7041)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7003..7022
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7042..7119)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7325..7344
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7432..7450
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggcagcagc catcaccatc atcaccacag ccagggtaaa ttattactga
      121 tattaggtag tgttattgca cttcctacat ttgctgcagg tggtggtgac cttgatgcta
      181 gtgattacac tggtgtttct ttttggttag ttactgctgc tttattagca tctactgtat
      241 ttttctttgt tgaaagagat agagtttctg caaaatggaa aacatcatta actgtatctg
      301 gtcttgttac tggtattgct ttctggcatt acatgtacat gagaggggta tggattgaaa
      361 ctggtgattc gccaactgta tttagataca ttaattggtt actaacagtt cctctattaa
      421 tatgtgaatt ctacttaatt cttgctgctg caactaatgt tgctggatca ttatttaaga
      481 aattactagt tggttctctt gttatgcttg tgtttggtta catgggtgaa gcaggaatca
      541 tggctgcatg gcctgcattc attattgggt gtttagcttg ggtatacatg atttatgaat
      601 tatgggctgg agaaggaaaa tctgcatgta atactgcaag tcctgctgtg caatcagctt
      661 acaacacaat gatgtatatt atcatctttg gttgggcgat ttatcctgta ggttatttca
      721 caggttacct gatgggtgac ggtggatcag ctcttaactt aaaccttatc tataaccttg
      781 ctgactttgt taacaagatt ctatttggtt taattatatg gaatgttgct gttaaagaat
      841 cttctaatgc ttgaggccgc ataatgctta agtcgaacag aaagtaatcg tattgtacac
      901 ggccgcataa tcgaaattaa taacagaaag taatcgtatt gtacacggcc gcataatcga
      961 aattaatacg actcactata ggggaattgt gagcggataa caattcccca tcttagtata
     1021 ttagttaagt ataagaagga gatatacata tgggtatggc tagcatgact ggtggacagc
     1081 aaatgggtcg ggatctgtac gacgatgacg ataaggatct cgccaccatg gtcgactcat
     1141 cacgtcgtaa gtggaataag acaggtcacg cagtcagagc tataggtcgg ctgagctcac
     1201 tcgagaacgt ctatatcaag gccgacaagc agaagaacgg catcaaggcg aacttcaaga
     1261 tccgccacaa catcgaggac ggcggcgtgc agctcgccta ccactaccag cagaacaccc
     1321 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcgtg cagtccaaac
     1381 tttcgaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1441 ccgggatcac tctcggcatg gacgagctgt acaagggcgg taccggaggg agcatggtga
     1501 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1561 taaacggcca caagttcagc gtgtccggcg agggtgaggg cgatgccacc tacggcaagc
     1621 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1681 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     1741 acttcttcaa gtccgccatg cccgaaggct acatccagga gcgcaccatc ttcttcaagg
     1801 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1861 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1921 agtacaacct gccggaccaa ctgactgaag agcagatcgc agaatttaaa gaggaattct
     1981 ccctatttga caaggacggg gatgggacaa taacaaccaa ggagctgggg acggtgatgc
     2041 ggtctctggg gcagaacccc acagaagcag agctgcagga catgatcaat gaagtagatg
     2101 ccgacggtga cggcacaatc gacttccctg agttcctgac aatgatggca agaaaaatga
     2161 aatacaggga cacggaagaa gaaattagag aagcgttcgg tgtgtttgat aaggatggca
     2221 atggctacat cagtgcagca gagcttcgcc acgtgatgac aaaccttgga gagaagttaa
     2281 cagatgaaga ggttgatgaa atgatcaggg aagcagacat cgatggggat ggtcaggtaa
     2341 actacgaaga gtttgtacaa atgatgacag cgaagtgagg taccctcgag tctggtaaag
     2401 aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat
     2461 taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     2521 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     2581 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2641 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2701 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     2761 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2821 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2881 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2941 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3001 gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca
     3061 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3121 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3181 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3241 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3301 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3361 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3421 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3481 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3541 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3601 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3661 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3721 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3781 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3841 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3901 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3961 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4021 caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4081 tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4141 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4201 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4261 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4321 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4381 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4441 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4501 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4561 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4621 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4681 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4741 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4801 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4861 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4921 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     4981 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     5041 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     5101 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5161 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5221 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     5281 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     5341 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     5401 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     5461 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     5521 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     5581 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     5641 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     5701 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     5761 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     5821 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
     5881 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     5941 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     6001 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     6061 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     6121 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     6181 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     6241 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     6301 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     6361 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     6421 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     6481 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     6541 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     6601 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     6661 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     6721 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     6781 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     6841 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     6901 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     6961 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     7021 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     7081 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     7141 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     7201 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     7261 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     7321 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     7381 ccacgatgcg tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac
     7441 tcactatagg
//
