LOCUS       sequence-487441-        6009 bp    DNA     circular SYN 29-SEP-2025
DEFINITION  Production of N-terminally His-SUMO-tagged and C-terminally 
            Avi-tagged SH2 domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6009)
  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 6009)
  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: "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-SUMO-SHP2-C-SH2(E139D)-Avi
FEATURES             Location/Qualifiers
     source          1..6009
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    4..28
                     /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             43..65
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             85..102
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             112..129
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             136..429
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             778..822
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             854..871
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(924..942)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      938..985
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1022..1477
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1109..1128)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1319..1340
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1569..2384)
                     /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(1569..2384)
                     /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     2292..2311
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2376..2395
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2506..3094
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2995..3014
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3248..3265
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3280..3419
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3345..3364)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3505..3527
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3521..3712)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4521..5603)
                     /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     5565..5584
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5604..5681)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5887..5906
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5994..6009,1..4)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5994..6009,1..3)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
       61 ggagatatac atatgggcag cagccatcat catcatcatc acggcagcgg cctggtgccg
      121 cgcggcagcg ctagcatgtc ggactcagaa gtcaatcaag aagctaagcc agaggtcaag
      181 ccagaagtca agcctgagac tcacatcaat ttaaaggtgt ccgatggatc ttcagagatc
      241 ttcttcaaga tcaaaaagac cactccttta agaaggctga tggaagcgtt cgctaaaaga
      301 cagggtaagg aaatggactc cttaagattc ttgtacgacg gtattagaat tcaagctgat
      361 cagacccctg aagatttgga catggaggat aacgatatta ttgaggctca cagagaacag
      421 attggtggtg cagatcctac ctctgaaagg tggtttcatg gacatctctc tgggaaagaa
      481 gcagagaaat tattaactga aaaaggaaaa catggtagtt ttcttgtacg agatagccag
      541 agccaccctg gagattttgt tctttctgtg cgcactggtg atgacaaagg ggagagcaat
      601 gacggcaagt ctaaagtgac ccatgttatg attcgctgtc aggaactgaa atacgacgtt
      661 ggtggaggag aacggtttga ttctttgaca gatcttgtgg aacattataa gaagaatcct
      721 atggtggaaa cattgggtac agtactacaa ctcaagcagc cccttaacac gactcgtggc
      781 ctgaacgata tttttgaagc gcagaaaatt gaatggcatg aataacgtcg acaagcttgc
      841 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
      901 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
      961 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     1021 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1081 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1141 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1201 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     1261 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     1321 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     1381 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     1441 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     1501 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1561 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     1621 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     1681 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     1741 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     1801 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     1861 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     1921 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     1981 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     2041 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     2101 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     2161 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     2221 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     2281 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     2341 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     2401 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     2461 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2521 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2581 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2641 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2701 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2761 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2821 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2881 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2941 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3001 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3061 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3121 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3181 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3241 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3301 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     3361 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     3421 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3481 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3541 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     3601 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     3661 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     3721 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     3781 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     3841 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     3901 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     3961 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4021 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4081 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4141 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4201 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4261 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4321 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     4381 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     4441 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4501 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4561 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4621 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     4681 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     4741 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     4801 atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     4861 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     4921 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     4981 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5041 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5101 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5161 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5221 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5281 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5341 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     5401 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     5461 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     5521 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     5581 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     5641 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     5701 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     5761 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     5821 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     5881 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     5941 ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga aattaatacg
     6001 actcactat
//
