LOCUS       sequence-428821-        6542 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CapN-caged TEVcs on yeast surface.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6542)
  AUTHORS   Shen J, Geng L, Li X, Emery C, Kroning K, Shingles G, Lee K, Heyden 
            M, Li P, Wang W
  TITLE     A general method for chemogenetic control of peptide function.
  JOURNAL   Nat Methods. 2023 Jan;20(1):112-122. doi: 
            10.1038/s41592-022-01697-8. Epub 2022 Dec 8.
  PUBMED    36481965
REFERENCE   2  (bases 1 to 6542)
  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"; doi: 
            "10.1038/s41592-022-01697-8"; journalName: "Nat Methods"; date: 
            "2023-01"; volume: "20"; issue: "1"; pages: "112-122"
COMMENT     Sequence Label: pCTCON2-Aga2p-Flag-CapN-TEVcs-HA
FEATURES             Location/Qualifiers
     source          1..6542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    69..572
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        609..713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(714..782,783..1574)
                     /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     complement(932..951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1745..2333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2234..2253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2487..2504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2621..2642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2657..2662,2663..2680,2681..2687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2695..2711
                     /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)."
     primer_bind     2700..2722
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2719..2735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2719..2735
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2756..2774
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2804..3456
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3008..3125
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3420..3443
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             3775..3786
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3877..3900
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3907..4227
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             4270..4290
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4297..4323
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(4656..4675)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4657..4675)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4682..4699)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4682..4698)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4691..4713)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4839..5294
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4926..4945)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5136..5157
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5397..6071)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(6072..6352)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(6373..6392)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6492..6514
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1861 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     2641 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctcgaaatta
     2761 accctcacta aagggaacaa aagctggtac caattccttg aattttcaaa aattcttact
     2821 ttttttttgg atggacgcaa agaagtttaa taatcatatt acatggcatt accaccatat
     2881 acatatccat atctaatctt acttatatgt tgtggaaatg taaagagccc cattatctta
     2941 gcctaaaaaa accttctctt tggaactttc agtaatacgc ttaactgctc attgctatat
     3001 tgaagtacgg attagaagcc gccgagcggg tgacagccct ccgaaggaag actctcctcc
     3061 gtgcgtcctc gtcttcaccg gtcgcgttcc tgaaacgcag atgtgcctcg cgccgcactg
     3121 ctccgaacaa taaagattct acaatactag cttttatggt tatgaagagg aaaaattggc
     3181 agtaacctgg ccccacaaac cttcaaatga acgaatcaaa ttaacaacca taggatgata
     3241 atgcgattag ttttttagcc ttatttctgg ggtaattaat cagcgaagcg atgatttttg
     3301 atctattaac agatatataa atgcaaaaac tgcataacca ctttaactaa tactttcaac
     3361 attttcggtt tgtattactt cttattcaaa tgtaataaaa gtatcaacaa aaaattgtta
     3421 atatacctct atactttaac gtcaaggaga aaaaaccccg gatcgaattc cctacttcat
     3481 acattttcaa ttaagatgca gttacttcgc tgtttttcaa tattttctgt tattgcttca
     3541 gttttagcac aggaactgac aactatatgc gagcaaatcc cctcaccaac tttagaatcg
     3601 acgccgtact ctttgtcaac gactactatt ttggccaacg ggaaggcaat gcaaggagtt
     3661 tttgaatatt acaaatcagt aacgtttgtc agtaattgcg gttctcaccc ctcaacaact
     3721 agcaaaggca gccccataaa cacacagtat gtttttaagg acaatagctc gacgattgaa
     3781 ggtagaggag gttctggtag cggtaacgac tctacagacg gtagcgctct gcagcaaggt
     3841 ctgcaggcta gtggtggagg aggctctggt gctagcgact acaaggatga cgacgataag
     3901 actagtggag tgcaggtgga aaccatctcc ccaggagacg ggcgcacctt ccccaagcgc
     3961 ggccagacct gcgtggtgca ctacaccggg atgcttgaag atggaaagaa agttgattcc
     4021 tcccgggaca gaaacaagcc ctttaagttt atgctaggca agcaggaggt gatccgaggc
     4081 tgggaagaag gggttgccca gatgagtgtg ggtcagagag ccaaactgac tatatctcca
     4141 gattatgcct atggtgccac tgggcaccca ggcatcatcc caccacatgc cactctcgtc
     4201 ttcgatgtgg agcttctaaa actggaaacg cgtggagtgg aggaagtggc gcggtatagt
     4261 cctaatttgg aaaatctcta cttccagggt ggatcctacc catatgacgt tccagattac
     4321 gcttaatagc tcgagatctg ataacaacag tgtagatgta acaaaatcga ctttgttccc
     4381 actgtacttt tagctcgtac aaaatacaat atacttttca tttctccgta aacaacatgt
     4441 tttcccatgt aatatccttt tctatttttc gttccgttac caactttaca catactttat
     4501 atagctattc acttctatac actaaaaaac taagacaatt ttaattttgc tgcctgccat
     4561 atttcaattt gttataaatt cctataattt atcctattag tagctaaaaa aagatgaatg
     4621 tgaatcgaat cctaagagaa ttgagctcca attcgcccta tagtgagtcg tattacaatt
     4681 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     4741 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     4801 gcctttccca acagttgcgc agcctgaatg gcgaatggac gcgccctgta gcggcgcatt
     4861 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4921 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4981 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5041 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5101 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5161 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5221 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5281 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     5341 cgcatagatc ggcaagtgca caaacaatac ttaaataaat actactcagt aataacctat
     5401 ttcttagcat ttttgacgaa atttgctatt ttgttagagt cttttacacc atttgtctcc
     5461 acacctccgc ttacatcaac accaataacg ccatttaatc taagcgcatc accaacattt
     5521 tctggcgtca gtccaccagc taacataaaa tgtaagcttt cggggctctc ttgccttcca
     5581 acccagtcag aaatcgagtt ccaatccaaa agttcacctg tcccacctgc ttctgaatca
     5641 aacaagggaa taaacgaatg aggtttctgt gaagctgcac tgagtagtat gttgcagtct
     5701 tttggaaata cgagtctttt aataactggc aaaccgagga actcttggta ttcttgccac
     5761 gactcatctc catgcagttg gacgatatca atgccgtaat cattgaccag agccaaaaca
     5821 tcctccttag gttgattacg aaacacgcca accaagtatt tcggagtgcc tgaactattt
     5881 ttatatgctt ttacaagact tgaaattttc cttgcaataa ccgggtcaat tgttctcttt
     5941 ctattgggca cacatataat acccagcaag tcagcatcgg aatctagagc acattctgcg
     6001 gcctctgtgc tctgcaagcc gcaaactttc accaatggac cagaactacc tgtgaaatta
     6061 ataacagaca tactccaagc tgcctttgtg tgcttaatca cgtatactca cgtgctcaat
     6121 agtcaccaat gccctccctc ttggccctct ccttttcttt tttcgaccga attaattctt
     6181 aatcggcaaa aaaagaaaag ctccggatca agattgtacg taaggtgaca agctattttt
     6241 caataaagaa tatcttccac tactgccatc tggcgtcata actgcaaagt acacatatat
     6301 tacgatgctg ttctattaaa tgcttcctat attatatata tagtaatgtc gtgatctatg
     6361 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     6421 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     6481 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     6541 ga
//
