LOCUS       sequence-146045-        6887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of double-cysteine variant of Malonyl-coenzyme A 
            synthetase, RpMatB, as scaffold for ATP biosensor - tighter binding.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6887)
  AUTHORS   Vancraenenbroeck R, Kunzelmann S, Webb MR
  TITLE     Development of a range of fluorescent reagentless biosensors for 
            ATP, based on malonyl-coenzyme A synthetase.
  JOURNAL   PLoS One. 2017 Jun 21;12(6):e0179547. doi: 
            10.1371/journal.pone.0179547. eCollection 2017.
  PUBMED    28636641
REFERENCE   2  (bases 1 to 6887)
  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: "PLoS One.";
            date: "2017-06-21"; volume: "12(6):e0179547. doi"; pages: " 
            10.1371/journal.pone.0179547. eCollection 2017"
COMMENT     Sequence Label: pTEV5_ RpMatB_2
FEATURES             Location/Qualifiers
     source          1..6887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             18..35
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             57..77
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1606..1624)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1620..1667
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1704..2159
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1791..1810)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2001..2022
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2186..2290
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2291..2359,2360..3151)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2509..2528)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     3805..3824
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4058..4075
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4090..4232
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4158..4177)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4318..4340
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4334..4525)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5334..6416)
                     /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     6378..6397
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6417..6494)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6700..6719
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6803..6822
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6803..6821
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6822..6846
                     /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             6861..6883
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 catatatgtc gtactaccat caccatcacc atcacgatta cgatatccca actagtgaaa
       61 acctgtattt tcagggcgct agcatgaacg ccaacctgtt cgcccgcctg ttcgataagc
      121 tcgacgaccc ccacaagctc gcgatcgaaa ccgcggccgg ggacaagatc agctacgccg
      181 agctggtggc gcgggcgggc cgcgtcgcca acgtgctggt ggcacgcggc ctgcaggtcg
      241 gcgaccgcgt tgcggcgcaa accgagaagt cggtggaagc gctggtgctg tatctcgcca
      301 cggtgcgggc cggcggcgtg tatctgccgc tcaacaccgc ctatacgctg cacgagctcg
      361 attacttcat caccgatgcc gagccgaaga tcgtggtggc cgatccgtcc aagcgcgacg
      421 ggatcgcggc gattgccgcc aaggtcggcg ccacggtgga gacgcttggc cccgacggtc
      481 ggggctcgct caccgatgcg gcagctggag ccagcgaggc gttcgccacg atcgaccgcg
      541 gcgccgatga tctggcggcg atcctctaca cctcagggac gaccggccgc gccaagggcg
      601 cgatgctcag ccacgacaat ttggcgtcga actcgctgac gctggtcgat tactggcgct
      661 tcacgccgga tgacgtgctg atccacgcgc tgccgatcta tcacacccat ggattgttcg
      721 tggccagcaa cgtcacgctg ttcgcgcgcg gatcgatgat cttcctgccg aagttcgatc
      781 ccgacaagat cctcgacctg atggcgcgcg ccaccgtgct gatgggtgtg ccgacgttct
      841 acacgcggct cttgcagagc ccgcggctga ccaaggagac gacgggccac atgaggctgt
      901 tcatctccgg gtcggcgccg ctgctcgccg atacgcattg cgaatggtcg gcgaagaccg
      961 gtcacgccgt gctcgagcgc tacggcatga ccgagaccaa catgaacacc tcgaacccgt
     1021 atgacggcga ccgcgtcccc ggcgcggtcg gcccggcgct gcccggcgtt tcggcgcgcg
     1081 tgaccgatcc ggaaaccggc aaggaactgc cgcgcggcga catcgggatg atcgaggtga
     1141 agggcccgaa cgtgttcaag ggctactggc ggatgccgga gaagaccaag tctgaattcc
     1201 gcgacgacgg cttcttcatc accggcgacc tcggcaagat cgacgagcgc ggctacgtcc
     1261 acatcctcgg ccgcggcaag gatctggtga tcaccggcgg cttcaacgtc tatccgaagg
     1321 aaatcgagag cgagatcgac gccatgccgg gcgtggtcga atccgcggtg atcggcgtgc
     1381 cgcacgccga tttcggcgag ggcgtcactg ccgtggtggt gcgcgacaag ggtgccacga
     1441 tcgacgaagc gtgcgtgctg cacggcctcg acggtcagct cgccaagttc aagatgccga
     1501 agaaagtgat cttcgtcgac gacctgccgc gcaacaccat gggtgcggtc cagaagaacg
     1561 tcctgcgcga gacctacaag gacatctaca agtaaggatc cgcggccgct gagcaataac
     1621 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1681 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1741 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1801 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1861 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1921 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1981 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2041 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2101 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
     2161 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2221 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2281 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2341 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2401 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2461 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2521 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2581 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2641 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2701 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     2761 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     2821 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     2881 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     2941 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3001 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3061 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3121 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3181 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3241 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3301 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3361 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3421 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3481 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3541 aatcctgtta ccagctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3601 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3661 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3721 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3781 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3841 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3901 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3961 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4021 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4081 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     4141 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     4201 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     4261 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4321 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     4381 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     4441 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     4501 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     4561 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4621 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4681 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4741 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4801 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4861 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4921 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4981 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     5041 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     5101 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     5161 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     5221 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     5281 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     5341 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5401 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     5461 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     5521 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     5581 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     5641 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5701 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5761 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     5821 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5881 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5941 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     6001 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     6061 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     6121 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     6181 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     6241 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     6301 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     6361 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     6421 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     6481 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     6541 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     6601 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     6661 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     6721 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     6781 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     6841 aattcccctc tagaaataat tttgtttaac tttaagaagg agatata
//
