LOCUS       sequence-387343-        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(G41)
FEATURES             Location/Qualifiers
     source          1..7816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     35..54
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        35..53
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    54..78
                     /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             93..115
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             131..154
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             161..175
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             185..1069
                     /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             2846..2875
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(2898..2916)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2912..2959
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2996..3451
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3083..3102)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3293..3314
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3566..4357,4358..4426))
                     /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     4189..4208
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4427..4519)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4600..5188
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5089..5108
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5342..5359
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5374..5513
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5439..5458)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5599..5621
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5615..5806)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6378..7460)
                     /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     7422..7441
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7461..7538)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7744..7763
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tagaggatcg agatcgatct cgatcccgcg aaattaatac gactcactat aggggaattg
       61 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
      121 catggcaagc tggagccacc cgcagttcga aaagggtgca gatgacgacg acaaggtacc
      181 gcatgaaatc ggtactggct ttccattcga cccccattat gtggaagtcc tgggcgagcg
      241 catgcactac gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa
      301 cccgacctcc tcctacgtgt ggcgcaacat catcccgcat gttgcaccga cccatcgctg
      361 cattgctcca gacctgatcg gtatgggcaa atccgacaaa ccagacctgg gttatttctt
      421 cgacgaccac gtccgcttca tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt
      481 cctggtcatt cacgactggg gctccgctct gggtttccac tgggccaagc gcaatccaga
      541 gcgcgtcaaa ggtattgcat ttatggagtt catccgccct atcccgacct gggacgaatg
      601 gccagaattt gcccgcgaga ccttccaggc cttccgcacc accgacgtcg gccgcaagct
      661 gatcatcgat cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct
      721 gactgaagtc gagatggacc attaccgcga gccgttcctg aatcctgttg accgcgagcc
      781 actgtggcgc ttcccaaacg agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct
      841 ggtcgaagaa tacatggact ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg
      901 caccccaggc gttctgatcc caccggccga agccgctcgc ctggccaaaa gcctgcctaa
      961 ctgcaaggct gtggacatcg gcccgggtct gaatctgctg caagaagaca acccggacct
     1021 gatcggcagc gagatcgcgc gctggctgtc tactctggag atttccggtg gcagcctggc
     1081 agaagcggca gccaaagagg cggctgccaa agaagctgcc gccaaggagg cagcagctaa
     1141 agaagccgca gctaaagcag cggctggcgg cagcgttagc aaaggtgaag aactgtttac
     1201 cggcgttgtg ccgattctgg tggaactgga tggtgatgtg aatggccata aatttagcgt
     1261 tcgtggcgaa ggcgaaggtg atgcgaccaa cggtaaactg accctgaaat ttatttgcac
     1321 caccggtaaa ctgccggttc cgtggccgac cctggtgacc accctgacct atggcgttca
     1381 gtgctttagc cgctatccgg atcatatgaa acgccatgat ttctttaaaa gcgcgatgcc
     1441 ggaaggctat gtgcaggaac gtaccattag cttcaaagat gatggcacct ataaaacccg
     1501 tgcggaagtt aaatttgaag gcgataccct ggtgaaccgc attgaactga aaggtattga
     1561 ttttaaagaa gatggcaaca ttctgggtca taaactggaa tataatttca acagccataa
     1621 cgtgtatatt accgccgata aacagaaaaa tggcatcaaa gcgaacttta aaatccgtca
     1681 caacgtggaa gatggtagcg tgcagctggc ggatcattat cagcagaata ccccgattgg
     1741 tgatggcccg gtgctgctgc cggataatca ttatctgagc acccagagcg ttctgagcaa
     1801 agatccgaat gaaaaacgtg atcatatggt gctgctggaa tttgttaccg ccgcgggcat
     1861 tacccacggt ggatccccgc atcatgtttt tgttagcgat tttgtggatt ttagcatcta
     1921 tgttgatgca ccggaagatc tgctgcagac ctggtatatc aatcgctttc tgaaatttcg
     1981 tgaaggtgca tataccgatc cggatagcta ttttcataac tatgccaaac tgaccaaaga
     2041 agaggcaatt aaaaccgcaa tgacctggtg gaaagaaatt aactggctga atctgaaaca
     2101 gaacattctg ccgacacgtg aacgtgcaag cctgattctg accaaaagcg caaatcatgc
     2161 agttgaagaa gttcgtctgc gtaaaggctc tggcggtacc ggtaccccgt atctgcagtt
     2221 tgatcgtaat cagtgggcag cactgcgtga tagcgttccg atgacactga gcgaagatga
     2281 aattgcacgt ctgaaaggca ttaatgaaga tctgagcctg gaagaagttg ccgaaattta
     2341 tctgccgctg agccgtctgc tgaatttcta tatttcaagc aatctgcgtc gtcaggcagt
     2401 tctggaacag tttctgggca ccaatggtca gcgtattccg tatattatca gcattgcagg
     2461 tagcgttgca gttggtaaaa gcaccaccgc acgtgttctg caggcactgc tgagtcgttg
     2521 gcctgaacat cgtcgtgttg aactgattac caccgcgggt tttctgcatc cgaatcaggt
     2581 tctgaaagaa cgtggtctga tgaaaaaaaa gggtttcccg gaaagctatg atatgcatcg
     2641 tctggttaaa tttgtgagcg atctgaaaag cggtgttccg aatgttaccg caccggttta
     2701 tagccatctg atttatgatg ttattccgga tggtgataaa accgttgttc agccggatat
     2761 tctgattctg gaaggtctga atgtgctgca gagcggtatg gattatccgc atgatggagc
     2821 tccgggcttc tcctcaattt ccgctcatca ccaccatcat caccatcacc accactaatt
     2881 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     2941 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     3001 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3061 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3121 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3181 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3241 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3301 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3361 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3421 aattttaaca aaatattaac gtttacaatt tctggcggca cgatggcatg agattatcaa
     3481 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3541 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3601 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3661 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3721 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3781 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3841 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3901 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3961 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4021 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4081 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4141 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4201 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4261 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4321 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4381 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4441 aatcatgatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4501 atttagaaaa ataaacaaat aggtcatgac caaaatccct taacgtgagt tttcgttcca
     4561 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4621 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4681 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4741 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4801 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4861 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4921 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4981 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5041 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5101 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5161 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5221 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5281 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5341 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     5401 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     5461 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     5521 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5581 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5641 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     5701 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     5761 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     5821 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     5881 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     5941 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     6001 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     6061 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     6121 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     6181 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     6241 tcaacgacag gagcacgatc atgctagtca tgccccgcgc ccaccggaag gagctgactg
     6301 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     6361 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     6421 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     6481 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     6541 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     6601 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg
     6661 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     6721 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     6781 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     6841 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     6901 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     6961 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     7021 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     7081 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     7141 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     7201 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     7261 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     7321 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     7381 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     7441 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     7501 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     7561 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     7621 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     7681 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     7741 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     7801 cacgatgcgt ccggcg
//
