LOCUS       sequence-487428-        6916 bp    DNA     circular SYN 29-SEP-2025
DEFINITION  Expression of the kinase domain of c-Src in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6916)
  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 6916)
  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 c-Src KD
FEATURES             Location/Qualifiers
     source          1..6916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      139..326
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(194..218)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(449..468)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(450..468)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(478..495)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(478..494)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(487..509)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      635..1090
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(722..741)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     932..953
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1150..1365
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             1366..2169
                     /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(2316..2335)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2435..2457
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2495..2513)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    2555..3058
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        3095..3199
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3200..3268,3269..4060)
                     /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(3418..3437)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4231..4819
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4720..4739
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4973..4990
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5107..5128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5143..5148,5149..5166,5167..5173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5181..5197
                     /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     5186..5208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5205..5221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5205..5221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5240..5260
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5242..5260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5344..6007
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    5560..5677
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     5972..5995
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             6041..6058
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6080..6100
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
ORIGIN
        1 caagctttgg acttcttcgc cagaggtttg gtcaagtctc caatcaaggt tgtcggcttg
       61 tctaccttgc cagaaattta cgaaaagatg gaaaagggtc aaatcgttgg tagatacgtt
      121 gttgacactt ctaaataagc gaatttctta tgatttatga tttttattat taaataagtt
      181 ataaaaaaaa taagtgtata caaattttaa agtgactctt aggttttaaa acgaaaattc
      241 ttattcttga gtaactcttt cctgtaggtc aggttgcttt ctcaggtata gcatgaggtc
      301 gctcttattg accacacctc taccggcatg caccattcct tgcggcggcg gtgctcaacg
      361 gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgac
      421 ctcgaggggg ggcccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
      481 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
      541 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
      601 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc
      661 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
      721 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
      781 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
      841 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
      901 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
      961 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1021 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1081 cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     1141 tatcgacggt tcaattcatc attttttttt tattcttttt tttgatttcg gtttccttga
     1201 aatttttttg attcggtaat ctccgaacag aaggaagaac gaaggaagga gcacagactt
     1261 agattggtat atatacgcat atgtagtgtt gaagaaacat gaaattgccc agtattctta
     1321 acccaactgc acagaacaaa aacctgcagg aaacgaagat aaatcatgtc gaaagctaca
     1381 tataaggaac gtgctgctac tcatcctagt cctgttgctg ccaagctatt taatatcatg
     1441 cacgaaaagc aaacaaactt gtgtgcttca ttggatgttc gtaccaccaa ggaattactg
     1501 gagttagttg aagcattagg tcccaaaatt tgtttactaa aaacacatgt ggatatcttg
     1561 actgattttt ccatggaggg cacagttaag ccgctaaagg cattatccgc caagtacaat
     1621 tttttactct tcgaagacag aaaatttgct gacattggta atacagtcaa attgcagtac
     1681 tctgcgggtg tatacagaat agcagaatgg gcagacatta cgaatgcaca cggtgtggtg
     1741 ggcccaggta ttgttagcgg tttgaagcag gcggcagaag aagtaacaaa ggaacctaga
     1801 ggccttttga tgttagcaga attgtcatgc aagggctccc tatctactgg agaatatact
     1861 aagggtactg ttgacattgc gaagagcgac aaagattttg ttatcggctt tattgctcaa
     1921 agagacatgg gtggaagaga tgaaggttac gattggttga ttatgacacc cggtgtgggt
     1981 ttagatgaca agggagacgc attgggtcaa cagtatagaa ccgtggatga tgtggtctct
     2041 acaggatctg acattattat tgttggaaga ggactatttg caaagggaag ggatgctaag
     2101 gtagagggtg aacgttacag aaaagcaggc tgggaagcat atttgagaag atgcggccag
     2161 caaaactaaa aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct
     2221 tcaatttaat tatatcagtt attaccctat gcggtgtgaa ataccgcaca gatgcgtaag
     2281 gagaaaatac cgcatcaggt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     2341 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2401 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     2461 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     2521 gttaatgtca tgataataat ggtttcttag gacggatcgc ttgcctgtaa cttacacgcg
     2581 cctcgtatct tttaatgatg gaataatttg ggaatttact ctgtgtttat ttatttttat
     2641 gttttgtatt tggattttag aaagtaaata aagaaggtag aagagttacg gaatgaagaa
     2701 aaaaaaataa acaaaggttt aaaaaatttc aacaaaaagc gtactttaca tatatattta
     2761 ttagacaaga aaagcagatt aaatagatat acattcgatt aacgataagt aaaatgtaaa
     2821 atcacaggat tttcgtgtgt ggtcttctac acagacaaga tgaaacaatt cggcattaat
     2881 acctgagagc aggaagagca agataaaagg tagtatttgt tggcgatccc cctagagtct
     2941 tttacatctt cggaaaacaa aaactatttt ttctttaatt tcttttttta ctttctattt
     3001 ttaatttata tatttatatt aaaaaattta aattataatt atttttatag cacgtgatga
     3061 aaaggaccca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3121 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3181 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3241 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3301 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3361 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3421 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3481 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3541 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3601 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3661 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3721 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3781 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3841 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3901 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3961 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4021 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4081 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4141 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4201 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4261 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4321 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4381 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4441 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4501 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4561 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4621 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4681 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4741 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4801 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4861 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4921 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4981 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     5041 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     5101 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     5161 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     5221 ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctcca
     5281 ccgcggtggc ggccggccgc tctcgcgaca cgaggccctt tcgtcttcaa gaattccttg
     5341 aattttcaaa aattcttact ttttttttgg atggacgcaa agaagtttaa taatcatatt
     5401 acatggcatt accaccatat acatatccat atacatatcc atatctaatc ttacttatat
     5461 gttgtggaaa tgtaaagagc cccattatct tagcctaaaa aaaccttctc tttggaactt
     5521 tcagtaatac gcttaactgc tcattgctat attgaagtac ggattagaag ccgccgagcg
     5581 ggtgacagcc ctccgaagga agactctcct ccgtgcgtcc tcgtcttcac cggtcgcgtt
     5641 cctgaaacgc agatgtgcct cgcgccgcac tgctccgaac aataaagatt ctacaatact
     5701 agcttttatg gttatgaaga ggaaaaattg gcagtaacct ggccccacaa accttcaaat
     5761 gaacgaatca aattaacaac cataggatga taatgcgatt agttttttag ccttatttct
     5821 ggggtaatta atcagcgaag cgatgatttt tgatctatta acagatatat aaatgcaaaa
     5881 actgcataac cactttaact aatactttca acattttcgg tttgtattac ttcttattca
     5941 aatgtaataa aagtatcaac aaaaaattgt taatatacct ctatacttta acgtcaagga
     6001 gaaaaaaacc ccggatccgg aattccagat gggcagcagc catcatcatc atcatcacga
     6061 ttacgatatc ccaacgaccg aaaaccttta cttccagggc catatgaagg atgcctggga
     6121 gatccctcgg gagtcgctgc ggctggaggt caagctgggc cagggctgct ttggcgaggt
     6181 gtggatgggg acctggaacg gtaccaccag ggtggccatc aaaaccctga agcctggcac
     6241 gatgtctcca gaggccttcc tgcaggaggc ccaggtcatg aagaagctga ggcatgagaa
     6301 gctggtgcag ttgtatgctg tggtttcaga ggagcccatt tacatcgtca cggagtacat
     6361 gagcaagggg agtttgctgg actttctcaa gggggagaca ggcaagtacc tgcggctgcc
     6421 tcagctggtg gacatggctg ctcagatcgc ctcaggcatg gcgtacgtgg agcggatgaa
     6481 ctacgtccac cgggaccttc gtgcagccaa catcctggtg ggagagaacc tggtgtgcaa
     6541 agtggccgac tttgggctgg ctcggctcat tgaagacaat gagtacacgg cgcggcaagg
     6601 tgccaaattc cccatcaagt ggacggctcc agaagctgcc ctctatggcc gcttcaccat
     6661 caagtcggac gtgtggtcct tcgggatcct gctgactgag ctcaccacaa agggacgggt
     6721 gccctaccct gggatggtga accgcgaggt gctggaccag gtggagcggg gctaccggat
     6781 gccctgcccg ccggagtgtc ccgagtccct gcacgacctc atgtgccagt gctggcggaa
     6841 ggagcctgag gagcggccca ccttcgagta cctgcaggcc ttcctggagg actacttcac
     6901 gtccaccgag ccctag
//
