LOCUS       sequence-267452-        7361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast expression of uTEV3 fused to mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7361)
  AUTHORS   Sanchez MI, Ting AY
  TITLE     Directed evolution improves the catalytic efficiency of TEV 
            protease.
  JOURNAL   Nat Methods. 2019 Dec 9. pii: 10.1038/s41592-019-0665-7. doi: 
            10.1038/s41592-019-0665-7.
  PUBMED    31819267
REFERENCE   2  (bases 1 to 7361)
  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 
            Methods. 2019 Dec 9. pii: 10.1038/s41592-019-0665-7. doi: 
            10.1038/s41592-019-0665-7."
COMMENT     Sequence Label: Gal:mCherry-uTEV3
FEATURES             Location/Qualifiers
     source          1..7361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      66..75
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             72..779
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(217..235)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     485..504
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             885..1592
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGTFWKHWIQTKDGQCGNPLVSTRDGFI
                     VGIHSASNFTNTNNYFASVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     terminator      1623..1870
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1639..1657)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1888..1907)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1889..1907)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1917..1934)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1917..1933)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1926..1948)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2074..2529
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2161..2180)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2371..2392
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2789..3451)
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(3452..3639)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     primer_bind     complement(3786..3805)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3905..3927
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3965..3983)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4024..4527
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4564..4668
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4669..4737,4738..5529)
                     /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(4887..4906)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5700..6288
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6189..6208
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6442..6459
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6576..6597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6612..6617,6618..6635,6636..6642)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6650..6666
                     /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     6674..6690
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6674..6690
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6709..6729
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6711..6729
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6920..7361
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    6920..7037
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     7332..7355
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
ORIGIN
        1 aaaaaacccc ggatcgaatt ctacttcata cattttcaat taagatggct cgggatccac
       61 cggtcgccac catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
      121 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
      181 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
      241 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
      301 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
      361 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
      421 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
      481 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
      541 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
      601 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
      661 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
      721 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagg
      781 ctctgggagg tcaaggtctg caggctagtg gtggaggagg ctctggtgga ggcggtagcg
      841 gaggcggagg gtcggctagc ggcagcggaa agcttggttc cgggggagaa tccttgttta
      901 agggaccacg tgattacaac ccgatatcga gcaccatttg tcatttgacg aatgaatctg
      961 atgggcacac aacatcgttg tatggtattg gatttggtcc cttcatcatt acaaacaagc
     1021 acttgtttag aagaaataat ggaacactgt tggtccaatc actacatggt gtattcaagg
     1081 tcaagaacac cacgactttg caacaacacc tcattgatgg gagggacatg ataattattc
     1141 gcatgcctaa ggatttccca ccatttcctc aaaagctgaa atttagagag ccacaaaggg
     1201 aagagcgcat atgtcttgtg acaaccaact tccaaactaa gagcatgtct agcatggtgt
     1261 cagacactag ttgcacattc ccttcatctg atggcacatt ctggaagcat tggattcaaa
     1321 ccaaggatgg gcagtgtggc aatccattag tatcaactag agatgggttc attgttggta
     1381 tacactcagc atcgaatttc accaacacaa acaattattt cgcaagcgtg ccgaaaaact
     1441 tcatggaatt gttgacaaat caggaggcgc agcagtgggt tagtggttgg cgattaaatg
     1501 ctgactcagt attgtggggg ggccataaag ttttcatggt gaaacctgaa gagccttttc
     1561 agccagttaa ggaagcgact caactcatga atgaattggt ctacagccag taatagctcg
     1621 agtcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac
     1681 cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat
     1741 gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt
     1801 gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc
     1861 tttaatttgc ggccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     1921 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     1981 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     2041 tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg
     2101 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2161 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2221 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2281 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2341 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2401 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     2461 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     2521 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     2581 agatccgtcg agttcaagag aaaaaaaaag aaaaagcaaa aagaaaaaag gaaagcgcgc
     2641 ctcgttcaga atgacacgta tagaatgatg cattaccttg tcatcttcag tatcatactg
     2701 ttcgtataca tacttactga cattcatagg tatacatata tacacatgta tatatatcgt
     2761 atgctgcaac tttaaataat cggtgtcact acataagaac acctttggtg gagggaacat
     2821 cgtttgtacc attgggcgag gtggcttctc ttatggcaac cgcaagagcc ttgaacgcac
     2881 tctcactacg gtgatgatca ttcttgcctc gcagacaatc aacgtggagg gtaattctgc
     2941 tagcctctgc aaaactttca agaaaatgcg ggatcatctc gcaagagaga tctcctactt
     3001 tctccctttg caaaccaagt tcgacaactg cgtacggcct gttcgaaaga tctaccaccg
     3061 ctctggaaag tgcctcatcc aaaggcgcaa atcctgatcc aaaccttttt actccacgca
     3121 cggcccctag ggcctcttta aatgcttgac cgagagcaat cccgcagtct tcagtggtgt
     3181 gatggtcgtc tatgtgtaag tcaccaatgc actcaacgat tagcgaccag ccggaatgct
     3241 tggccagagc atgtatcata tggtccagaa accctatacc tgtgtggacg ttaatcactt
     3301 gcgattgtgt ggcctgttct gctactgctt ctgcctcttt ttctgggaag atcgagtgct
     3361 ctatcgctag gggaccaccc tttaaagaga tcgcaatctg aatcttggtt tcatttgtaa
     3421 tacgctttac tagggctttc tgctctgtca tctttgcctt cgtttatctt gcctgctcat
     3481 tttttagtat attcttcgaa gaaatcacat tactttatat aatgtataat tcattatgtg
     3541 ataatgccaa tcgctaagaa aaaaaaagag tcatccgcta ggtggaaaaa aaaaaatgaa
     3601 aatcattacc gaggcataaa aaaatataga gtgtactaga ggaggccaag agtaatagaa
     3661 aaagaaaatt gcgggaaagg actgtgttat gacttccctg actaatgccg tgttcaaacg
     3721 atacctggca gtgactccta gcgctcacca agctcttaaa acgggaattt atggtgcact
     3781 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     3841 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3901 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     3961 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4021 acggatcgct tgcctgtaac ttacacgcgc ctcgtatctt ttaatgatgg aataatttgg
     4081 gaatttactc tgtgtttatt tatttttatg ttttgtattt ggattttaga aagtaaataa
     4141 agaaggtaga agagttacgg aatgaagaaa aaaaaataaa caaaggttta aaaaatttca
     4201 acaaaaagcg tactttacat atatatttat tagacaagaa aagcagatta aatagatata
     4261 cattcgatta acgataagta aaatgtaaaa tcacaggatt ttcgtgtgtg gtcttctaca
     4321 cagacaagat gaaacaattc ggcattaata cctgagagca ggaagagcaa gataaaaggt
     4381 agtatttgtt ggcgatcccc ctagagtctt ttacatcttc ggaaaacaaa aactattttt
     4441 tctttaattt ctttttttac tttctatttt taatttatat atttatatta aaaaatttaa
     4501 attataatta tttttatagc acgtgatgaa aaggacccag gtggcacttt tcggggaaat
     4561 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4621 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4681 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4741 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4801 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4861 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4921 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     4981 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5041 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5101 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5161 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5221 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5281 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5341 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5401 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5461 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5521 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5581 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5641 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5701 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5761 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5821 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5881 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5941 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6001 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6061 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6121 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6181 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6241 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6301 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6361 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6421 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     6481 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     6541 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     6601 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     6661 taacaatttc acgcaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct
     6721 cactaaaggg aacaaaagct ggagctccgc aaagaagttt aataatcata ttacatggca
     6781 ttaccaccat atacatatcc atatctaatc ttacttatat gttgtggaaa tgtaaagagc
     6841 cccattatct tagcctaaaa aaaccttctc tttggaactt tcagtaatac gcttaactgc
     6901 tcattgctat attgaagtac ggattagaag ccgccgagcg ggtgacagcc ctccgaagga
     6961 agactctcct ccgtgcgtcc tcgtcttcac cggtcgcgtt cctgaaacgc agatgtgcct
     7021 cgcgccgcac tgctccgaac aataaagatt ctacaatact agcttttatg gttatgaaga
     7081 ggaaaaattg gcagtaacct ggccccacaa accttcaaat gaacgaatca aattaacaac
     7141 cataggatga taatgcgatt agttttttag ccttatttct ggggtaatta atcagcgaag
     7201 cgatgatttt tgatctatta acagatatat aaatgcaaaa actgcataac cactttaact
     7261 aatactttca acattttcgg tttgtattac ttcttattca aatgtaataa aagtatcaac
     7321 aaaaaattgt taatatacct ctatacttta acgtcaagga g
//
