LOCUS       sequence-488719-        5979 bp    DNA     circular SYN 08-OCT-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 5979)
  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 5979)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 8, 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-N-SH2(K55R)-Avi
FEATURES             Location/Qualifiers
     source          1..5979
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..41
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        22..40
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    41..65
                     /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             80..102
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             122..139
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             149..166
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             173..466
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             785..829
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             861..878
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(931..949)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      945..992
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1029..1484
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1116..1135)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1326..1347
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1576..2391)
                     /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(1576..2391)
                     /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     2299..2318
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2383..2402
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2513..3101
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3002..3021
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3255..3272
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3287..3426
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3352..3371)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3512..3534
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3528..3719)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4528..5610)
                     /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     5572..5591
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5611..5688)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5894..5913
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tcgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
       61 attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata tgggcagcag
      121 ccatcatcat catcatcacg gcagcggcct ggtgccgcgc ggcagcgcta gcatgtcgga
      181 ctcagaagtc aatcaagaag ctaagccaga ggtcaagcca gaagtcaagc ctgagactca
      241 catcaattta aaggtgtccg atggatcttc agagatcttc ttcaagatca aaaagaccac
      301 tcctttaaga aggctgatgg aagcgttcgc taaaagacag ggtaaggaaa tggactcctt
      361 aagattcttg tacgacggta ttagaattca agctgatcag acccctgaag atttggacat
      421 ggaggataac gatattattg aggctcacag agaacagatt ggtggtatga catcgcggag
      481 atggtttcac ccaaatatca ctggtgtgga ggcagaaaac ctactgttga caagaggagt
      541 tgatggcagt tttttggcaa ggcctagtaa aagtaaccct ggagacttca cactttccgt
      601 tagaagaaat ggagctgtca cccacatcag gattcagaac actggtgatt actatgacct
      661 gtatggaggg gagaaatttg ccactttggc tgagttggtc cagtattaca tggaacatca
      721 cgggcaatta aaagagaaga atggagatgt cattgagctt aaatatcctc tgaactgtgc
      781 agatggcctg aacgatattt ttgaagcgca gaaaattgaa tggcatgaat aacgtcgaca
      841 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
      901 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
      961 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     1021 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1081 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     1141 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1201 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     1261 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1321 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     1381 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     1441 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     1501 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1561 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     1621 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     1681 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     1741 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     1801 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     1861 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     1921 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     1981 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     2041 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     2101 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     2161 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     2221 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     2281 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     2341 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     2401 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     2461 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2521 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2581 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2641 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2701 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2761 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2821 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2881 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2941 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3001 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3061 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3121 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3181 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3241 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3301 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     3361 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3421 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3481 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3541 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3601 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3661 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3721 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3781 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     3841 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     3901 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     3961 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     4021 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4081 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4141 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     4201 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     4261 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     4321 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     4381 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     4441 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     4501 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4561 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4621 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     4681 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     4741 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     4801 taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     4861 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     4921 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     4981 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     5041 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     5101 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5161 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5221 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5281 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     5341 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     5401 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     5461 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     5521 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     5581 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     5641 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     5701 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     5761 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     5821 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     5881 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     5941 tgatgccggc cacgatgcgt ccggcgtaga ggatcgaga
//
