LOCUS       sequence-488349-        7126 bp    DNA     circular SYN 03-OCT-2025
DEFINITION  Production of His-tagged full-length SHP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7126)
  AUTHORS   van Vlimmeren AE, Voleti R, Chartier CA, Jiang Z, Karandur D, 
            Humphries PA, Lo WL, Shah NH
  TITLE     The pathogenic T42A mutation in SHP2 rewires the interaction 
            specificity of its N-terminal regulatory domain.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Jul 23;121(30):e2407159121. doi: 
            10.1073/pnas.2407159121. Epub 2024 Jul 16.
  PUBMED    39012820
REFERENCE   2  (bases 1 to 7126)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 3, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2024-07-23"; volume: "121(30):e2407159121. 
            doi"; pages: " 10.1073/pnas.2407159121. Epub 2024 Jul 16"
COMMENT     Sequence Label: pET-28b His6-TEV-SHP2-FL(L43F)
FEATURES             Location/Qualifiers
     source          1..7126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     475..494
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     728..745
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    760..899
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(825..844)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     985..1007
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1001..1192)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2001..3083)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3045..3064
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3084..3161)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3367..3386
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3470..3489
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3470..3488
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3489..3513
                     /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)."
     RBS             3528..3550
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3569..3586
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3608..3628
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5460..5477
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5530..5548)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5544..5591
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5628..6083
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5715..5734)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5925..5946
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6175..6990)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6175..6990)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6898..6917
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6982..7001
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(7112..7126,1..574)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
       61 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
      121 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
      181 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
      241 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
      301 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
      361 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
      421 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
      481 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
      541 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
      601 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
      661 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
      721 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
      781 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
      841 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
      901 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
      961 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     1021 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     1081 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     1141 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     1201 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     1261 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     1321 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     1381 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     1441 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     1501 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     1561 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     1621 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     1681 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     1741 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     1801 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     1861 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     1921 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     1981 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2041 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     2101 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     2161 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     2221 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     2281 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     2341 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     2401 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     2461 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     2521 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     2581 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     2641 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     2701 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     2761 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     2821 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     2881 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     2941 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     3001 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     3061 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     3121 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     3181 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     3241 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     3301 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     3361 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     3421 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     3481 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     3541 aagaaggaga tataccatgg gcagcagcca tcatcatcat catcacgatt acgatatccc
     3601 aacgaccgaa aacctttact tccagggcca tatgacatcg cggagatggt ttcacccaaa
     3661 tatcactggt gtggaggcag aaaacctact gttgacaaga ggagttgatg gcagtttttt
     3721 ggcaaggcct agtaaaagta accctggaga cttcacattt tccgttagaa gaaatggagc
     3781 tgtcacccac atcaagattc agaacactgg tgattactat gacctgtatg gaggggagaa
     3841 atttgccact ttggctgagt tggtccagta ttacatggaa catcacgggc aattaaaaga
     3901 gaagaatgga gatgtcattg agcttaaata tcctctgaac tgtgcagatc ctacctctga
     3961 aaggtggttt catggacatc tctctgggaa agaagcagag aaattattaa ctgaaaaagg
     4021 aaaacatggt agttttcttg tacgagagag ccagagccac cctggagatt ttgttctttc
     4081 tgtgcgcact ggtgatgaca aaggggagag caatgacggc aagtctaaag tgacccatgt
     4141 tatgattcgc tgtcaggaac tgaaatacga cgttggtgga ggagaacggt ttgattcttt
     4201 gacagatctt gtggaacatt ataagaagaa tcctatggtg gaaacattgg gtacagtact
     4261 acaactcaag cagcccctta acacgactcg tataaatgct gctgaaatag aaagcagagt
     4321 tcgagaacta agcaaattag ctgagaccac agataaagtc aaacaaggct tttgggaaga
     4381 atttgagaca ctacaacaac aggagtgcaa acttctctac agccgaaaag agggtcaaag
     4441 gcaagaaaac aaaaacaaaa atagatataa aaacatcctg ccctttgatc ataccagggt
     4501 tgtcctacac gatggtgatc ccaatgagcc tgtttcagat tacatcaatg caaatatcat
     4561 catgcctgaa tttgaaacca agtgcaacaa ttcaaagccc aaaaagagtt acattgccac
     4621 acaaggctgc ctgcaaaaca cggtgaatga cttttggcgg atggtgttcc aagaaaactc
     4681 ccgagtgatt gtcatgacaa cgaaagaagt ggagagagga aagagtaaat gtgtcaaata
     4741 ctggcctgat gagtatgctc taaaagaata tggcgtcatg cgtgttagga acgtcaaaga
     4801 aagcgccgct catgactata cgctaagaga acttaaactt tcaaaggttg gacaagggaa
     4861 tacggagaga acggtctggc aataccactt tcggacctgg ccggaccacg gcgtgcccag
     4921 cgaccctggg ggcgtgctgg acttcctgga ggaggtgcac cataagcagg agagcatcat
     4981 ggatgcaggg ccggtcgtgg tgcactgcag tgctggaatt ggccggacag ggacgttcat
     5041 tgtgattgat attcttattg acatcatcag agagaaaggt gttgactgcg atattgacgt
     5101 tcccaaaacc atccagatgg tgcggtctca gaggtcaggg atggtccaga cagaagcaca
     5161 gtaccgattt atctatatgg cggtccagca ttatattgaa acactacagc gcaggattga
     5221 agaagagcag aaaagcaaga ggaaagggca cgaatataca aatattaagt attctctagc
     5281 ggaccagacg agtggagatc agagccctct cccgccttgt actccaacgc caccctgtgc
     5341 agaaatgaga gaagacagtg ctagagtcta tgaaaacgtg ggcctgatgc aacagcagaa
     5401 aagtttcaga tagggatccg aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc
     5461 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     5521 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     5581 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     5641 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5701 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5761 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5821 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     5881 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5941 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     6001 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     6061 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     6121 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     6181 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     6241 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     6301 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     6361 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     6421 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     6481 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     6541 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     6601 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     6661 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     6721 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     6781 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     6841 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     6901 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     6961 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     7021 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     7081 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttt
//
