LOCUS       sequence-387349-        7758 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 7758)
  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 7758)
  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/TO_CoA-Snifit(V97T)
FEATURES             Location/Qualifiers
     source          1..7758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(82..101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        273..652
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        653..856
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     807..827
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    858..876
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    879..897
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     907..931
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1028..1037
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1034..1924
                     /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(2030..2032,2033..2545)
                     /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(2072..2093)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2680..2701
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3748..3765)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3754..3978
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4111..4130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4262..4309
                     /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             4317..5339
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4317..5339
                     /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    5469..5590
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5506..5525)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5560..5579
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5639..5655)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5639..5655)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5652..5674)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5663..5679
                     /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(5687..5693,5694..5711,5712..5717))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5732..5753
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5870..5887)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6041..6629)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6121..6140)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6800..7591,7592..7660))
                     /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     7423..7442
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7661..7758,1..7))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
       61 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
      121 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
      181 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
      241 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
      301 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      361 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
      421 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      481 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
      541 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
      601 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
      661 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
      721 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
      781 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
      841 ctatataagc agagctctcc ctatcagtga tagagatctc cctatcagtg atagagatcg
      901 tcgacgagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
      961 cctccataga agacaccggg accgatccag cctccggact ctagcgttta aacttaagct
     1021 tggtaccgcc accatggcag agatcggaac cggctttcct ttcgaccctc actacgtgga
     1081 agtgctgggc gagagaatgc actatgtgga cgtgggcccc agagatggaa cccctgtgct
     1141 gtttctgcac ggcaacccta ccagcagcta cgtgtggcgg aacatcatcc ctcacgtggc
     1201 ccctacacac agatgtatcg cccctgacct gatcggcatg ggcaagagcg ataagcccga
     1261 cctgggctac ttcttcgacg accacgtgcg gttcatggac gcctttattg aggccctcgg
     1321 cctggaagag gtggtgctgg ttattcacga ttggggcagc gccctgggat tccactgggc
     1381 caagagaaac cccgagagag tgaagggaat cgccttcatg gaattcatca ggcccattcc
     1441 tacctgggac gagtggcctg agttcgccag agagacattc caggccttca gaaccaccga
     1501 cgtgggcaga aagctgatca tcgaccagaa cgtgttcatc gagggcaccc tgcctatggg
     1561 agtcgttaga cctctgaccg aggtggaaat ggaccactac agagagccct ttctgaaccc
     1621 cgtggaccgg gaacctcttt ggagattccc caacgagctg cccattgccg gcgagcctgc
     1681 taatattgtg gccctggtgg aagagtacat ggactggctg catcagagcc ccgtgcctaa
     1741 gctgctgttt tggggaacac ccggcgtgct gattcctcct gctgaagctg ccagactggc
     1801 caagagcctg cctaattgca aggccgtgga tatcggccct ggcctgaatc tgctgcaaga
     1861 ggacaacccc gatctgatcg gctccgagat tgccagatgg ctgagcaccc tggaaatcag
     1921 cggaggatct ctggctgaag ccgccgctaa agaagccgct gcaaaagagg ctgccgccaa
     1981 agaggcagca gcaaaagaag cagccgctaa ggctgctgct ggcggatctg tgagcaaggg
     2041 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     2101 ccacaagttc agcgtgcgcg gcgagggcga gggcgatgcc accaacggca agctgaccct
     2161 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     2221 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcgcc acgacttctt
     2281 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcagcttca aggacgacgg
     2341 cacctacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     2401 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     2461 cttcaacagc cacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa
     2521 cttcaagatc cgccacaacg tggaggacgg cagcgtgcag ctcgccgacc actaccagca
     2581 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     2641 gtccgtgctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     2701 gaccgccgcc gggatcactc acggcggatc ccctcaccac gtgttcgtgt ccgacttcgt
     2761 ggacttcagc atctacgtgg acgcccctga ggatctgctg cagacctggt acatcaaccg
     2821 gtttctgaag ttcagagagg gcgcctacac agaccccgac agctacttcc acaactacgc
     2881 caagctgacc aaagaggaag ccatcaagac cgccatgacc tggtggaaag agatcaactg
     2941 gctgaacctg aagcagaaca tcctgcctac cagagagcgg gccagcctga tcctgaccaa
     3001 atctgccaat cacgccgtgg aagaagtgcg gctgagaaaa ggatctggcg gcaccggcac
     3061 accctacctg cagttcgata gaaaccagtg ggccgctctg agagacagcg tgccaatgac
     3121 actgagcgag gacgagatcg ccagactgaa gggcatcaat gaggacctga gcctggaaga
     3181 ggtggccgag atttacctgc ccctgagccg gctgctgaac ttctacatca gcagcaacct
     3241 gcggagacag gccgtgctgg aacagttcct gggaacaaac ggccagcgga tcccctacat
     3301 catctctatc gccggatcta ccgccgtggg caagagcaca acagctagag ttctgcaggc
     3361 cctgctgagc agatggcccg agcatagaag agtggaactg attaccaccg ccggctttct
     3421 gcaccccaac caggtgctga aagaacgggg cctgatgaag aagaagggct tccccgagag
     3481 ctacgacatg caccggctgg tcaagtttgt gtccgacctg aagtccggcg tgcccaatgt
     3541 gacagcccct gtgtacagcc acctgatcta cgacgtgatc cccgacggcg ataagaccgt
     3601 ggtgcagccc gatatcctga ttctggaagg cctgaacgtg ctgcagagcg gcatggacta
     3661 tcctcatgat ggcgctccag gattctcctc aatttccgct taagcggccg ctcgagtcta
     3721 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     3781 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3841 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3901 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3961 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     4021 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4081 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4141 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4201 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct
     4261 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa
     4321 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     4381 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     4441 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     4501 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     4561 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     4621 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     4681 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     4741 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     4801 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     4861 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     4921 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     4981 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     5041 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     5101 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     5161 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     5221 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     5281 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     5341 acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5401 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5461 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5521 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5581 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     5641 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5701 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5761 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5821 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5881 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5941 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6001 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6061 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6121 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6181 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6241 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6301 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6361 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6421 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6481 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6541 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6601 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6661 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6721 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6781 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6841 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6901 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6961 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     7021 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     7081 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     7141 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     7201 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     7261 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7321 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7381 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7441 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7501 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7561 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7621 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7681 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7741 aaaataaaca aatagggg
//
