LOCUS       sequence-387298-        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(V97T)
FEATURES             Location/Qualifiers
     source          1..7691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        132..511
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        512..715
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     666..686
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     760..779
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        760..778
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      820..829
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             826..1716
                     /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(1822..1824,1825..2337)
                     /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(1864..1885)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2472..2493
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3540..3557)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3546..3770
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3903..3922)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4054..4101
                     /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             4109..5131
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4109..5131
                     /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    5261..5382
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5298..5317)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5352..5371
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5431..5447)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5431..5447)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5444..5466)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5455..5471
                     /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(5479..5485,5486..5503,5504..5509))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5524..5545
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5662..5679)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5833..6421)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5913..5932)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6592..7383,7384..7452))
                     /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     7215..7234
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7453..7557)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7632..7651)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
       61 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
      121 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
      181 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      241 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      301 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      361 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
      421 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      481 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
      541 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
      601 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
      661 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
      721 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
      781 gacccaagct ggctagcgtt taaacttaag cttggtaccg ccaccatggc agagatcgga
      841 accggctttc ctttcgaccc tcactacgtg gaagtgctgg gcgagagaat gcactatgtg
      901 gacgtgggcc ccagagatgg aacccctgtg ctgtttctgc acggcaaccc taccagcagc
      961 tacgtgtggc ggaacatcat ccctcacgtg gcccctacac acagatgtat cgcccctgac
     1021 ctgatcggca tgggcaagag cgataagccc gacctgggct acttcttcga cgaccacgtg
     1081 cggttcatgg acgcctttat tgaggccctc ggcctggaag aggtggtgct ggttattcac
     1141 gattggggca gcgccctggg attccactgg gccaagagaa accccgagag agtgaaggga
     1201 atcgccttca tggaattcat caggcccatt cctacctggg acgagtggcc tgagttcgcc
     1261 agagagacat tccaggcctt cagaaccacc gacgtgggca gaaagctgat catcgaccag
     1321 aacgtgttca tcgagggcac cctgcctatg ggagtcgtta gacctctgac cgaggtggaa
     1381 atggaccact acagagagcc ctttctgaac cccgtggacc gggaacctct ttggagattc
     1441 cccaacgagc tgcccattgc cggcgagcct gctaatattg tggccctggt ggaagagtac
     1501 atggactggc tgcatcagag ccccgtgcct aagctgctgt tttggggaac acccggcgtg
     1561 ctgattcctc ctgctgaagc tgccagactg gccaagagcc tgcctaattg caaggccgtg
     1621 gatatcggcc ctggcctgaa tctgctgcaa gaggacaacc ccgatctgat cggctccgag
     1681 attgccagat ggctgagcac cctggaaatc agcggaggat ctctggctga agccgccgct
     1741 aaagaagccg ctgcaaaaga ggctgccgcc aaagaggcag cagcaaaaga agcagccgct
     1801 aaggctgctg ctggcggatc tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1861 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgcg cggcgagggc
     1921 gagggcgatg ccaccaacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1981 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     2041 taccccgacc acatgaagcg ccacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2101 caggagcgca ccatcagctt caaggacgac ggcacctaca agacccgcgc cgaggtgaag
     2161 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2221 ggcaacatcc tggggcacaa gctggagtac aacttcaaca gccacaacgt ctatatcacc
     2281 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa cgtggaggac
     2341 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2401 ctgctgcccg acaaccacta cctgagcacc cagtccgtgc tgagcaaaga ccccaacgag
     2461 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tcacggcgga
     2521 tcccctcacc acgtgttcgt gtccgacttc gtggacttca gcatctacgt ggacgcccct
     2581 gaggatctgc tgcagacctg gtacatcaac cggtttctga agttcagaga gggcgcctac
     2641 acagaccccg acagctactt ccacaactac gccaagctga ccaaagagga agccatcaag
     2701 accgccatga cctggtggaa agagatcaac tggctgaacc tgaagcagaa catcctgcct
     2761 accagagagc gggccagcct gatcctgacc aaatctgcca atcacgccgt ggaagaagtg
     2821 cggctgagaa aaggatctgg cggcaccggc acaccctacc tgcagttcga tagaaaccag
     2881 tgggccgctc tgagagacag cgtgccaatg acactgagcg aggacgagat cgccagactg
     2941 aagggcatca atgaggacct gagcctggaa gaggtggccg agatttacct gcccctgagc
     3001 cggctgctga acttctacat cagcagcaac ctgcggagac aggccgtgct ggaacagttc
     3061 ctgggaacaa acggccagcg gatcccctac atcatctcta tcgccggatc taccgccgtg
     3121 ggcaagagca caacagctag agttctgcag gccctgctga gcagatggcc cgagcataga
     3181 agagtggaac tgattaccac cgccggcttt ctgcacccca accaggtgct gaaagaacgg
     3241 ggcctgatga agaagaaggg cttccccgag agctacgaca tgcaccggct ggtcaagttt
     3301 gtgtccgacc tgaagtccgg cgtgcccaat gtgacagccc ctgtgtacag ccacctgatc
     3361 tacgacgtga tccccgacgg cgataagacc gtggtgcagc ccgatatcct gattctggaa
     3421 ggcctgaacg tgctgcagag cggcatggac tatcctcatg atggcgctcc aggattctcc
     3481 tcaatttccg cttaagcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc
     3541 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3601 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     3661 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3721 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     3781 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     3841 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3901 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3961 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     4021 aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc
     4081 tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga
     4141 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
     4201 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     4261 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     4321 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc
     4381 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     4441 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     4501 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     4561 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     4621 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     4681 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     4741 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     4801 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     4861 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     4921 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     4981 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     5041 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     5101 ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc
     5161 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     5221 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5281 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5341 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     5401 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5461 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5521 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5581 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5641 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5701 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5761 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5821 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5881 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5941 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6001 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6061 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6121 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6181 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6241 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6301 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6361 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6421 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6481 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6541 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6601 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6661 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6721 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6781 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6841 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6901 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6961 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7021 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7081 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7141 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7201 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7261 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7321 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7381 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7441 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7501 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7561 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     7621 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     7681 tgtgtgttgg a
//
