LOCUS       sequence-387347-        7691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CoA-Snifit in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7691)
  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 7691)
  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: pCDNA5/FRT_CoA-Snifit(G41)
FEATURES             Location/Qualifiers
     source          1..7691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(105..124)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        296..675
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        676..879
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     830..850
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     924..943
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        924..942
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      984..993
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             990..1880
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             join(1986..1988,1989..2501)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVE"
     primer_bind     complement(2028..2049)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2636..2657
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3704..3721)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3710..3934
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4067..4086)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4218..4265
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4273..5295
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4273..5295
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5425..5546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5462..5481)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5516..5535
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5595..5611)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5595..5611)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5608..5630)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5619..5635
                     /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)."
     promoter        complement(join(5643..5649,5650..5667,5668..5673))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5688..5709
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5826..5843)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5997..6585)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6077..6096)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6756..7547,7548..7616))
                     /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     7379..7398
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7617..7691,1..30))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
       61 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
      121 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
      181 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
      241 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat
      301 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
      361 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      421 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      481 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      541 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      601 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      661 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
      721 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
      781 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
      841 ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc
      901 actgcttact ggcttatcga aattaatacg actcactata gggagaccca agctggctag
      961 cgtttaaact taagcttggt accgccacca tggcagagat cggaaccggc tttcctttcg
     1021 accctcacta cgtggaagtg ctgggcgaga gaatgcacta tgtggacgtg ggccccagag
     1081 atggaacccc tgtgctgttt ctgcacggca accctaccag cagctacgtg tggcggaaca
     1141 tcatccctca cgtggcccct acacacagat gtatcgcccc tgacctgatc ggcatgggca
     1201 agagcgataa gcccgacctg ggctacttct tcgacgacca cgtgcggttc atggacgcct
     1261 ttattgaggc cctcggcctg gaagaggtgg tgctggttat tcacgattgg ggcagcgccc
     1321 tgggattcca ctgggccaag agaaaccccg agagagtgaa gggaatcgcc ttcatggaat
     1381 tcatcaggcc cattcctacc tgggacgagt ggcctgagtt cgccagagag acattccagg
     1441 ccttcagaac caccgacgtg ggcagaaagc tgatcatcga ccagaacgtg ttcatcgagg
     1501 gcaccctgcc tatgggagtc gttagacctc tgaccgaggt ggaaatggac cactacagag
     1561 agccctttct gaaccccgtg gaccgggaac ctctttggag attccccaac gagctgccca
     1621 ttgccggcga gcctgctaat attgtggccc tggtggaaga gtacatggac tggctgcatc
     1681 agagccccgt gcctaagctg ctgttttggg gaacacccgg cgtgctgatt cctcctgctg
     1741 aagctgccag actggccaag agcctgccta attgcaaggc cgtggatatc ggccctggcc
     1801 tgaatctgct gcaagaggac aaccccgatc tgatcggctc cgagattgcc agatggctga
     1861 gcaccctgga aatcagcgga ggatctctgg ctgaagccgc cgctaaagaa gccgctgcaa
     1921 aagaggctgc cgccaaagag gcagcagcaa aagaagcagc cgctaaggct gctgctggcg
     1981 gatctgtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     2041 acggcgacgt aaacggccac aagttcagcg tgcgcggcga gggcgagggc gatgccacca
     2101 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     2161 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     2221 agcgccacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatca
     2281 gcttcaagga cgacggcacc tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2341 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2401 acaagctgga gtacaacttc aacagccaca acgtctatat caccgccgac aagcagaaga
     2461 acggcatcaa ggccaacttc aagatccgcc acaacgtgga ggacggcagc gtgcagctcg
     2521 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2581 actacctgag cacccagtcc gtgctgagca aagaccccaa cgagaagcgc gatcacatgg
     2641 tcctgctgga gttcgtgacc gccgccggga tcactcacgg cggatcccct caccacgtgt
     2701 tcgtgtccga cttcgtggac ttcagcatct acgtggacgc ccctgaggat ctgctgcaga
     2761 cctggtacat caaccggttt ctgaagttca gagagggcgc ctacacagac cccgacagct
     2821 acttccacaa ctacgccaag ctgaccaaag aggaagccat caagaccgcc atgacctggt
     2881 ggaaagagat caactggctg aacctgaagc agaacatcct gcctaccaga gagcgggcca
     2941 gcctgatcct gaccaaatct gccaatcacg ccgtggaaga agtgcggctg agaaaaggat
     3001 ctggcggcac cggcacaccc tacctgcagt tcgatagaaa ccagtgggcc gctctgagag
     3061 acagcgtgcc aatgacactg agcgaggacg agatcgccag actgaagggc atcaatgagg
     3121 acctgagcct ggaagaggtg gccgagattt acctgcccct gagccggctg ctgaacttct
     3181 acatcagcag caacctgcgg agacaggccg tgctggaaca gttcctggga acaaacggcc
     3241 agcggatccc ctacatcatc tctatcgccg gatctgtggc cgtgggcaag agcacaacag
     3301 ctagagttct gcaggccctg ctgagcagat ggcccgagca tagaagagtg gaactgatta
     3361 ccaccgccgg ctttctgcac cccaaccagg tgctgaaaga acggggcctg atgaagaaga
     3421 agggcttccc cgagagctac gacatgcacc ggctggtcaa gtttgtgtcc gacctgaagt
     3481 ccggcgtgcc caatgtgaca gcccctgtgt acagccacct gatctacgac gtgatccccg
     3541 acggcgataa gaccgtggtg cagcccgata tcctgattct ggaaggcctg aacgtgctgc
     3601 agagcggcat ggactatcct catgatggcg ctccaggatt ctcctcaatt tccgcttaag
     3661 cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     3721 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3781 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3841 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3901 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     3961 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4021 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     4081 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4141 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     4201 gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga
     4261 acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     4321 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     4381 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     4441 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     4501 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     4561 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     4621 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     4681 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     4741 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     4801 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     4861 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     4921 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     4981 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     5041 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     5101 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     5161 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     5221 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     5281 agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg
     5341 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5401 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     5461 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5521 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     5581 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5641 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     5701 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5761 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5821 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5881 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5941 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     6001 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     6061 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     6121 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6181 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6241 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     6301 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6361 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6421 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6481 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6541 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6601 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6661 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6721 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6781 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6841 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6901 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6961 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     7021 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     7081 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     7141 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7201 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7261 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     7321 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     7381 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7441 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7501 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7561 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7621 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7681 tttgaatgta t
//
