LOCUS       sequence-387346-        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(G41)
FEATURES             Location/Qualifiers
     source          1..7758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(90..109)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        281..660
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        661..864
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     815..835
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    866..884
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    887..905
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     915..939
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1036..1045
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1042..1932
                     /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(2038..2040,2041..2553)
                     /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(2080..2101)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2688..2709
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3756..3773)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3762..3986
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4119..4138)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4270..4317
                     /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             4325..5347
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4325..5347
                     /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    5477..5598
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5514..5533)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5568..5587
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5647..5663)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5647..5663)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5660..5682)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5671..5687
                     /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(5695..5701,5702..5719,5720..5725))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5740..5761
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5878..5895)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6049..6637)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6129..6148)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6808..7599,7600..7668))
                     /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     7431..7450
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7669..7758,1..15))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
       61 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
      121 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
      181 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
      241 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
      301 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      361 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      421 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      481 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      541 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      601 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      661 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      721 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      781 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      841 tgggaggtct atataagcag agctctccct atcagtgata gagatctccc tatcagtgat
      901 agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
      961 tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa
     1021 cttaagcttg gtaccgccac catggcagag atcggaaccg gctttccttt cgaccctcac
     1081 tacgtggaag tgctgggcga gagaatgcac tatgtggacg tgggccccag agatggaacc
     1141 cctgtgctgt ttctgcacgg caaccctacc agcagctacg tgtggcggaa catcatccct
     1201 cacgtggccc ctacacacag atgtatcgcc cctgacctga tcggcatggg caagagcgat
     1261 aagcccgacc tgggctactt cttcgacgac cacgtgcggt tcatggacgc ctttattgag
     1321 gccctcggcc tggaagaggt ggtgctggtt attcacgatt ggggcagcgc cctgggattc
     1381 cactgggcca agagaaaccc cgagagagtg aagggaatcg ccttcatgga attcatcagg
     1441 cccattccta cctgggacga gtggcctgag ttcgccagag agacattcca ggccttcaga
     1501 accaccgacg tgggcagaaa gctgatcatc gaccagaacg tgttcatcga gggcaccctg
     1561 cctatgggag tcgttagacc tctgaccgag gtggaaatgg accactacag agagcccttt
     1621 ctgaaccccg tggaccggga acctctttgg agattcccca acgagctgcc cattgccggc
     1681 gagcctgcta atattgtggc cctggtggaa gagtacatgg actggctgca tcagagcccc
     1741 gtgcctaagc tgctgttttg gggaacaccc ggcgtgctga ttcctcctgc tgaagctgcc
     1801 agactggcca agagcctgcc taattgcaag gccgtggata tcggccctgg cctgaatctg
     1861 ctgcaagagg acaaccccga tctgatcggc tccgagattg ccagatggct gagcaccctg
     1921 gaaatcagcg gaggatctct ggctgaagcc gccgctaaag aagccgctgc aaaagaggct
     1981 gccgccaaag aggcagcagc aaaagaagca gccgctaagg ctgctgctgg cggatctgtg
     2041 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     2101 gtaaacggcc acaagttcag cgtgcgcggc gagggcgagg gcgatgccac caacggcaag
     2161 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     2221 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcgccac
     2281 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cagcttcaag
     2341 gacgacggca cctacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     2401 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     2461 gagtacaact tcaacagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc
     2521 aaggccaact tcaagatccg ccacaacgtg gaggacggca gcgtgcagct cgccgaccac
     2581 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2641 agcacccagt ccgtgctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     2701 gagttcgtga ccgccgccgg gatcactcac ggcggatccc ctcaccacgt gttcgtgtcc
     2761 gacttcgtgg acttcagcat ctacgtggac gcccctgagg atctgctgca gacctggtac
     2821 atcaaccggt ttctgaagtt cagagagggc gcctacacag accccgacag ctacttccac
     2881 aactacgcca agctgaccaa agaggaagcc atcaagaccg ccatgacctg gtggaaagag
     2941 atcaactggc tgaacctgaa gcagaacatc ctgcctacca gagagcgggc cagcctgatc
     3001 ctgaccaaat ctgccaatca cgccgtggaa gaagtgcggc tgagaaaagg atctggcggc
     3061 accggcacac cctacctgca gttcgataga aaccagtggg ccgctctgag agacagcgtg
     3121 ccaatgacac tgagcgagga cgagatcgcc agactgaagg gcatcaatga ggacctgagc
     3181 ctggaagagg tggccgagat ttacctgccc ctgagccggc tgctgaactt ctacatcagc
     3241 agcaacctgc ggagacaggc cgtgctggaa cagttcctgg gaacaaacgg ccagcggatc
     3301 ccctacatca tctctatcgc cggatctgtg gccgtgggca agagcacaac agctagagtt
     3361 ctgcaggccc tgctgagcag atggcccgag catagaagag tggaactgat taccaccgcc
     3421 ggctttctgc accccaacca ggtgctgaaa gaacggggcc tgatgaagaa gaagggcttc
     3481 cccgagagct acgacatgca ccggctggtc aagtttgtgt ccgacctgaa gtccggcgtg
     3541 cccaatgtga cagcccctgt gtacagccac ctgatctacg acgtgatccc cgacggcgat
     3601 aagaccgtgg tgcagcccga tatcctgatt ctggaaggcc tgaacgtgct gcagagcggc
     3661 atggactatc ctcatgatgg cgctccagga ttctcctcaa tttccgctta agcggccgct
     3721 cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     3781 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3841 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3901 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3961 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     4021 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4081 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4141 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4201 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4261 acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt
     4321 ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
     4381 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
     4441 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
     4501 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
     4561 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
     4621 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga
     4681 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
     4741 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
     4801 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
     4861 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
     4921 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
     4981 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
     5041 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
     5101 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
     5161 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
     5221 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
     5281 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
     5341 ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     5401 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     5461 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5521 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5581 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     5641 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5701 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5761 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5821 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5881 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5941 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     6001 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6061 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6121 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     6181 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     6241 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     6301 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6361 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6421 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6481 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6541 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6601 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6661 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6721 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6781 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6841 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6901 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6961 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7021 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7081 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7141 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     7201 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7261 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7321 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7381 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7441 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7501 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7561 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7621 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7681 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7741 tatttagaaa aataaaca
//
