LOCUS       sequence-387342-        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(G41S)
FEATURES             Location/Qualifiers
     source          1..7816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     555..574
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(594..671)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     877..896
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     984..1003
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        984..1002
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1003..1027
                     /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             1042..1064
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1080..1103
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1110..1124
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             1134..2018
                     /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             3795..3824
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(3847..3865)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3861..3908
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3945..4400
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4032..4051)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4242..4263
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4515..5306,5307..5375))
                     /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     5138..5157
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5376..5468)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5549..6137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6038..6057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6291..6308
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6323..6462
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6388..6407)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6548..6570
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6564..6755)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7327..7816,1..593))
                     /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"
ORIGIN
        1 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
       61 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
      121 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
      181 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
      241 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
      301 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
      361 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
      421 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
      481 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
      541 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
      601 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
      661 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
      721 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
      781 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
      841 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
      901 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
      961 gatcgatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
     1021 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc atggcaagct
     1081 ggagccaccc gcagttcgaa aagggtgcag atgacgacga caaggtaccg catgaaatcg
     1141 gtactggctt tccattcgac ccccattatg tggaagtcct gggcgagcgc atgcactacg
     1201 tcgatgttgg tccgcgcgat ggcacccctg tgctgttcct gcacggtaac ccgacctcct
     1261 cctacgtgtg gcgcaacatc atcccgcatg ttgcaccgac ccatcgctgc attgctccag
     1321 acctgatcgg tatgggcaaa tccgacaaac cagacctggg ttatttcttc gacgaccacg
     1381 tccgcttcat ggatgccttc atcgaagccc tgggtctgga agaggtcgtc ctggtcattc
     1441 acgactgggg ctccgctctg ggtttccact gggccaagcg caatccagag cgcgtcaaag
     1501 gtattgcatt tatggagttc atccgcccta tcccgacctg ggacgaatgg ccagaatttg
     1561 cccgcgagac cttccaggcc ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc
     1621 agaacgtttt tatcgagggt acgctgccga tgggtgtcgt ccgcccgctg actgaagtcg
     1681 agatggacca ttaccgcgag ccgttcctga atcctgttga ccgcgagcca ctgtggcgct
     1741 tcccaaacga gctgccaatc gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat
     1801 acatggactg gctgcaccag tcccctgtcc cgaagctgct gttctggggc accccaggcg
     1861 ttctgatccc accggccgaa gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg
     1921 tggacatcgg cccgggtctg aatctgctgc aagaagacaa cccggacctg atcggcagcg
     1981 agatcgcgcg ctggctgtct actctggaga tttccggtgg cagcctggca gaagcggcag
     2041 ccaaagaggc ggctgccaaa gaagctgccg ccaaggaggc agcagctaaa gaagccgcag
     2101 ctaaagcagc ggctggcggc agcgttagca aaggtgaaga actgtttacc ggcgttgtgc
     2161 cgattctggt ggaactggat ggtgatgtga atggccataa atttagcgtt cgtggcgaag
     2221 gcgaaggtga tgcgaccaac ggtaaactga ccctgaaatt tatttgcacc accggtaaac
     2281 tgccggttcc gtggccgacc ctggtgacca ccctgaccta tggcgttcag tgctttagcc
     2341 gctatccgga tcatatgaaa cgccatgatt tctttaaaag cgcgatgccg gaaggctatg
     2401 tgcaggaacg taccattagc ttcaaagatg atggcaccta taaaacccgt gcggaagtta
     2461 aatttgaagg cgataccctg gtgaaccgca ttgaactgaa aggtattgat tttaaagaag
     2521 atggcaacat tctgggtcat aaactggaat ataatttcaa cagccataac gtgtatatta
     2581 ccgccgataa acagaaaaat ggcatcaaag cgaactttaa aatccgtcac aacgtggaag
     2641 atggtagcgt gcagctggcg gatcattatc agcagaatac cccgattggt gatggcccgg
     2701 tgctgctgcc ggataatcat tatctgagca cccagagcgt tctgagcaaa gatccgaatg
     2761 aaaaacgtga tcatatggtg ctgctggaat ttgttaccgc cgcgggcatt acccacggtg
     2821 gatccccgca tcatgttttt gttagcgatt ttgtggattt tagcatctat gttgatgcac
     2881 cggaagatct gctgcagacc tggtatatca atcgctttct gaaatttcgt gaaggtgcat
     2941 ataccgatcc ggatagctat tttcataact atgccaaact gaccaaagaa gaggcaatta
     3001 aaaccgcaat gacctggtgg aaagaaatta actggctgaa tctgaaacag aacattctgc
     3061 cgacacgtga acgtgcaagc ctgattctga ccaaaagcgc aaatcatgca gttgaagaag
     3121 ttcgtctgcg taaaggctct ggcggtaccg gtaccccgta tctgcagttt gatcgtaatc
     3181 agtgggcagc actgcgtgat agcgttccga tgacactgag cgaagatgaa attgcacgtc
     3241 tgaaaagcat taatgaagat ctgagcctgg aagaagttgc cgaaatttat ctgccgctga
     3301 gccgtctgct gaatttctat atttcaagca atctgcgtcg tcaggcagtt ctggaacagt
     3361 ttctgggcac caatggtcag cgtattccgt atattatcag cattgcaggt agcgttgcag
     3421 ttggtaaaag caccaccgca cgtgttctgc aggcactgct gagtcgttgg cctgaacatc
     3481 gtcgtgttga actgattacc accgcgggtt ttctgcatcc gaatcaggtt ctgaaagaac
     3541 gtggtctgat gaaaaaaaag ggtttcccgg aaagctatga tatgcatcgt ctggttaaat
     3601 ttgtgagcga tctgaaaagc ggtgttccga atgttaccgc accggtttat agccatctga
     3661 tttatgatgt tattccggat ggtgataaaa ccgttgttca gccggatatt ctgattctgg
     3721 aaggtctgaa tgtgctgcag agcggtatgg attatccgca tgatggagct ccgggcttct
     3781 cctcaatttc cgctcatcac caccatcatc accatcacca ccactaatta acctaggctg
     3841 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     3901 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     3961 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4021 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4081 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4141 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4201 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4261 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4321 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4381 aatattaacg tttacaattt ctggcggcac gatggcatga gattatcaaa aaggatcttc
     4441 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4501 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4561 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4621 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4681 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4741 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4801 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4861 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4921 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4981 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5041 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5101 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5161 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5221 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5281 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5341 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atcatgattg
     5401 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5461 taaacaaata ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5521 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5581 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5641 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     5701 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5761 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5821 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5881 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5941 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6001 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6061 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6121 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6181 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6241 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     6301 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     6361 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     6421 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     6481 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     6541 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     6601 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     6661 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     6721 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     6781 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     6841 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     6901 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     6961 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     7021 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     7081 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     7141 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     7201 agcacgatca tgctagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     7261 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     7321 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     7381 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     7441 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     7501 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     7561 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac
     7621 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     7681 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     7741 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     7801 tttatgccag ccagcc
//
