LOCUS       sequence-487425-        6061 bp    DNA     circular SYN 29-SEP-2025
DEFINITION  Protein expression in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6061)
  AUTHORS   Jiang Z, van Vlimmeren AE, Karandur D, Semmelman A, Shah NH
  TITLE     Deep mutational scanning of the multi-domain phosphatase SHP2 
            reveals mechanisms of regulation and pathogenicity.
  JOURNAL   Nat Commun. 2025 Jul 1;16(1):5464. doi: 10.1038/s41467-025-60641-4.
  PUBMED    40595497
REFERENCE   2  (bases 1 to 6061)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 29, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-07-1"; volume: "16(1):5464. doi"; pages: " 
            10.1038/s41467-025-60641-4"
COMMENT     Sequence Label: pWJ 1781 URA
FEATURES             Location/Qualifiers
     source          1..6061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      138..325
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(193..217)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(448..467)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(449..467)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(477..494)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(477..493)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(486..508)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      634..1089
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(721..740)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     931..952
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1149..1364
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             1365..2168
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     primer_bind     complement(2315..2334)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2434..2456
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2494..2512)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    2554..3057
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        3094..3198
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3199..3267,3268..4059)
                     /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(3417..3436)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4230..4818
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4719..4738
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4972..4989
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5106..5127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5142..5147,5148..5165,5166..5172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5180..5196
                     /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     5185..5207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5204..5220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5204..5220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5239..5259
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5241..5259
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5343..6007
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    5559..5676
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     5971..5994
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
ORIGIN
        1 aagctttgga cttcttcgcc agaggtttgg tcaagtctcc aatcaaggtt gtcggcttgt
       61 ctaccttgcc agaaatttac gaaaagatgg aaaagggtca aatcgttggt agatacgttg
      121 ttgacacttc taaataagcg aatttcttat gatttatgat ttttattatt aaataagtta
      181 taaaaaaaat aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct
      241 tattcttgag taactctttc ctgtaggtca ggttgctttc tcaggtatag catgaggtcg
      301 ctcttattga ccacacctct accggcatgc accattcctt gcggcggcgg tgctcaacgg
      361 cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgacc
      421 tcgagggggg gcccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
      481 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
      541 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
      601 tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg
      661 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      721 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
      781 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
      841 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
      901 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
      961 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1021 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1081 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     1141 atcgacggtt caattcatca tttttttttt attctttttt ttgatttcgg tttccttgaa
     1201 atttttttga ttcggtaatc tccgaacaga aggaagaacg aaggaaggag cacagactta
     1261 gattggtata tatacgcata tgtagtgttg aagaaacatg aaattgccca gtattcttaa
     1321 cccaactgca cagaacaaaa acctgcagga aacgaagata aatcatgtcg aaagctacat
     1381 ataaggaacg tgctgctact catcctagtc ctgttgctgc caagctattt aatatcatgc
     1441 acgaaaagca aacaaacttg tgtgcttcat tggatgttcg taccaccaag gaattactgg
     1501 agttagttga agcattaggt cccaaaattt gtttactaaa aacacatgtg gatatcttga
     1561 ctgatttttc catggagggc acagttaagc cgctaaaggc attatccgcc aagtacaatt
     1621 ttttactctt cgaagacaga aaatttgctg acattggtaa tacagtcaaa ttgcagtact
     1681 ctgcgggtgt atacagaata gcagaatggg cagacattac gaatgcacac ggtgtggtgg
     1741 gcccaggtat tgttagcggt ttgaagcagg cggcagaaga agtaacaaag gaacctagag
     1801 gccttttgat gttagcagaa ttgtcatgca agggctccct atctactgga gaatatacta
     1861 agggtactgt tgacattgcg aagagcgaca aagattttgt tatcggcttt attgctcaaa
     1921 gagacatggg tggaagagat gaaggttacg attggttgat tatgacaccc ggtgtgggtt
     1981 tagatgacaa gggagacgca ttgggtcaac agtatagaac cgtggatgat gtggtctcta
     2041 caggatctga cattattatt gttggaagag gactatttgc aaagggaagg gatgctaagg
     2101 tagagggtga acgttacaga aaagcaggct gggaagcata tttgagaaga tgcggccagc
     2161 aaaactaaaa aactgtatta taagtaaatg catgtatact aaactcacaa attagagctt
     2221 caatttaatt atatcagtta ttaccctatg cggtgtgaaa taccgcacag atgcgtaagg
     2281 agaaaatacc gcatcaggta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2341 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     2401 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     2461 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     2521 ttaatgtcat gataataatg gtttcttagg acggatcgct tgcctgtaac ttacacgcgc
     2581 ctcgtatctt ttaatgatgg aataatttgg gaatttactc tgtgtttatt tatttttatg
     2641 ttttgtattt ggattttaga aagtaaataa agaaggtaga agagttacgg aatgaagaaa
     2701 aaaaaataaa caaaggttta aaaaatttca acaaaaagcg tactttacat atatatttat
     2761 tagacaagaa aagcagatta aatagatata cattcgatta acgataagta aaatgtaaaa
     2821 tcacaggatt ttcgtgtgtg gtcttctaca cagacaagat gaaacaattc ggcattaata
     2881 cctgagagca ggaagagcaa gataaaaggt agtatttgtt ggcgatcccc ctagagtctt
     2941 ttacatcttc ggaaaacaaa aactattttt tctttaattt ctttttttac tttctatttt
     3001 taatttatat atttatatta aaaaatttaa attataatta tttttatagc acgtgatgaa
     3061 aaggacccag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3121 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3181 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     3241 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     3301 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     3361 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     3421 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     3481 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     3541 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     3601 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3661 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3721 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     3781 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3841 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3901 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     3961 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     4021 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     4081 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     4141 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4201 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4261 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4321 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     4381 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4441 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4501 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4561 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4621 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4681 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4741 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4801 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4861 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4921 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4981 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     5041 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     5101 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     5161 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     5221 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac
     5281 cgcggtggcg gccggccgct ctcgcgacac gaggcccttt cgtcttcaag aattccttga
     5341 attttcaaaa attcttactt tttttttgga tggacgcaaa gaagtttaat aatcatatta
     5401 catggcatta ccaccatata catatccata tacatatcca tatctaatct tacttatatg
     5461 ttgtggaaat gtaaagagcc ccattatctt agcctaaaaa aaccttctct ttggaacttt
     5521 cagtaatacg cttaactgct cattgctata ttgaagtacg gattagaagc cgccgagcgg
     5581 gtgacagccc tccgaaggaa gactctcctc cgtgcgtcct cgtcttcacc ggtcgcgttc
     5641 ctgaaacgca gatgtgcctc gcgccgcact gctccgaaca ataaagattc tacaatacta
     5701 gcttttatgg ttatgaagag gaaaaattgg cagtaacctg gccccacaaa ccttcaaatg
     5761 aacgaatcaa attaacaacc ataggatgat aatgcgatta gttttttagc cttatttctg
     5821 gggtaattaa tcagcgaagc gatgattttt gatctattaa cagatatata aatgcaaaaa
     5881 ctgcataacc actttaacta atactttcaa cattttcggt ttgtattact tcttattcaa
     5941 atgtaataaa agtatcaaca aaaaattgtt aatatacctc tatactttaa cgtcaaggag
     6001 aaaaaacccc ggatccggaa ttccagctga ccaccgttaa ccatggcaat tcccggggat
     6061 c
//
