LOCUS       sequence-387338-        7816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CoA-Snifit in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7816)
  AUTHORS   Xue L, Schnacke P, Frei MS, Koch B, Hiblot J, Wombacher R, Fabritz 
            S, Johnsson K
  TITLE     Probing coenzyme A homeostasis with semisynthetic biosensors.
  JOURNAL   Nat Chem Biol. 2022 Oct 31. doi: 10.1038/s41589-022-01172-7.
  PUBMED    36316571
REFERENCE   2  (bases 1 to 7816)
  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: "Nat Chem 
            Biol. 2022 Oct 31. doi: 10.1038/s41589-022-01172-7."
COMMENT     Sequence Label: pET51b(+)_CoA-Snifit(V97T)
FEATURES             Location/Qualifiers
     source          1..7816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..27
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     115..134
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        115..133
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    134..158
                     /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             173..195
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             211..234
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             241..255
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             265..1149
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             2926..2955
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(2978..2996)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2992..3039
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3076..3531
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3163..3182)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3373..3394
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3646..4437,4438..4506))
                     /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     4269..4288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4507..4599)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4680..5268
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5169..5188
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5422..5439
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5454..5593
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5519..5538)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5679..5701
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5695..5886)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6458..7540)
                     /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     7502..7521
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7541..7618)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
       61 cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg aaattaatac
      121 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
      181 actttaagaa ggagatatac catggcaagc tggagccacc cgcagttcga aaagggtgca
      241 gatgacgacg acaaggtacc gcatgaaatc ggtactggct ttccattcga cccccattat
      301 gtggaagtcc tgggcgagcg catgcactac gtcgatgttg gtccgcgcga tggcacccct
      361 gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt ggcgcaacat catcccgcat
      421 gttgcaccga cccatcgctg cattgctcca gacctgatcg gtatgggcaa atccgacaaa
      481 ccagacctgg gttatttctt cgacgaccac gtccgcttca tggatgcctt catcgaagcc
      541 ctgggtctgg aagaggtcgt cctggtcatt cacgactggg gctccgctct gggtttccac
      601 tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat ttatggagtt catccgccct
      661 atcccgacct gggacgaatg gccagaattt gcccgcgaga ccttccaggc cttccgcacc
      721 accgacgtcg gccgcaagct gatcatcgat cagaacgttt ttatcgaggg tacgctgccg
      781 atgggtgtcg tccgcccgct gactgaagtc gagatggacc attaccgcga gccgttcctg
      841 aatcctgttg accgcgagcc actgtggcgc ttcccaaacg agctgccaat cgccggtgag
      901 ccagcgaaca tcgtcgcgct ggtcgaagaa tacatggact ggctgcacca gtcccctgtc
      961 ccgaagctgc tgttctgggg caccccaggc gttctgatcc caccggccga agccgctcgc
     1021 ctggccaaaa gcctgcctaa ctgcaaggct gtggacatcg gcccgggtct gaatctgctg
     1081 caagaagaca acccggacct gatcggcagc gagatcgcgc gctggctgtc tactctggag
     1141 atttccggtg gcagcctggc agaagcggca gccaaagagg cggctgccaa agaagctgcc
     1201 gccaaggagg cagcagctaa agaagccgca gctaaagcag cggctggcgg cagcgttagc
     1261 aaaggtgaag aactgtttac cggcgttgtg ccgattctgg tggaactgga tggtgatgtg
     1321 aatggccata aatttagcgt tcgtggcgaa ggcgaaggtg atgcgaccaa cggtaaactg
     1381 accctgaaat ttatttgcac caccggtaaa ctgccggttc cgtggccgac cctggtgacc
     1441 accctgacct atggcgttca gtgctttagc cgctatccgg atcatatgaa acgccatgat
     1501 ttctttaaaa gcgcgatgcc ggaaggctat gtgcaggaac gtaccattag cttcaaagat
     1561 gatggcacct ataaaacccg tgcggaagtt aaatttgaag gcgataccct ggtgaaccgc
     1621 attgaactga aaggtattga ttttaaagaa gatggcaaca ttctgggtca taaactggaa
     1681 tataatttca acagccataa cgtgtatatt accgccgata aacagaaaaa tggcatcaaa
     1741 gcgaacttta aaatccgtca caacgtggaa gatggtagcg tgcagctggc ggatcattat
     1801 cagcagaata ccccgattgg tgatggcccg gtgctgctgc cggataatca ttatctgagc
     1861 acccagagcg ttctgagcaa agatccgaat gaaaaacgtg atcatatggt gctgctggaa
     1921 tttgttaccg ccgcgggcat tacccacggt ggatccccgc atcatgtttt tgttagcgat
     1981 tttgtggatt ttagcatcta tgttgatgca ccggaagatc tgctgcagac ctggtatatc
     2041 aatcgctttc tgaaatttcg tgaaggtgca tataccgatc cggatagcta ttttcataac
     2101 tatgccaaac tgaccaaaga agaggcaatt aaaaccgcaa tgacctggtg gaaagaaatt
     2161 aactggctga atctgaaaca gaacattctg ccgacacgtg aacgtgcaag cctgattctg
     2221 accaaaagcg caaatcatgc agttgaagaa gttcgtctgc gtaaaggctc tggcggtacc
     2281 ggtaccccgt atctgcagtt tgatcgtaat cagtgggcag cactgcgtga tagcgttccg
     2341 atgacactga gcgaagatga aattgcacgt ctgaaaggca ttaatgaaga tctgagcctg
     2401 gaagaagttg ccgaaattta tctgccgctg agccgtctgc tgaatttcta tatttcaagc
     2461 aatctgcgtc gtcaggcagt tctggaacag tttctgggca ccaatggtca gcgtattccg
     2521 tatattatca gcattgcagg tagcaccgca gttggtaaaa gcaccaccgc acgtgttctg
     2581 caggcactgc tgagtcgttg gcctgaacat cgtcgtgttg aactgattac caccgcgggt
     2641 tttctgcatc cgaatcaggt tctgaaagaa cgtggtctga tgaaaaaaaa gggtttcccg
     2701 gaaagctatg atatgcatcg tctggttaaa tttgtgagcg atctgaaaag cggtgttccg
     2761 aatgttaccg caccggttta tagccatctg atttatgatg ttattccgga tggtgataaa
     2821 accgttgttc agccggatat tctgattctg gaaggtctga atgtgctgca gagcggtatg
     2881 gattatccgc atgatggagc tccgggcttc tcctcaattt ccgctcatca ccaccatcat
     2941 caccatcacc accactaatt aacctaggct gctgccaccg ctgagcaata actagcataa
     3001 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     3061 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3121 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3181 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3241 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3301 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3361 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3421 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3481 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tctggcggca
     3541 cgatggcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3601 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3661 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3721 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3781 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3841 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3901 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3961 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4021 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4081 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4141 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4201 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4261 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4321 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4381 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4441 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4501 actcatactc ttcctttttc aatcatgatt gaagcattta tcagggttat tgtctcatga
     4561 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggtcatgac caaaatccct
     4621 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4681 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4741 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4801 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4861 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4921 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4981 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5041 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5101 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5161 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5221 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5281 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     5341 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     5401 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     5461 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     5521 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     5581 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     5641 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5701 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     5761 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     5821 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     5881 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     5941 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     6001 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     6061 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     6121 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     6181 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     6241 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     6301 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgctagtca tgccccgcgc
     6361 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     6421 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     6481 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6541 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     6601 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     6661 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     6721 cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     6781 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     6841 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     6901 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     6961 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     7021 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     7081 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     7141 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     7201 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     7261 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     7321 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     7381 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     7441 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     7501 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     7561 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     7621 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     7681 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     7741 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     7801 tggcgagccc gatctt
//
