LOCUS       sequence-488353-        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, Jiang Z, Karandur D, Applebaum Licht AT, Shah NH
  TITLE     The pathogenic E139D mutation stabilizes a non-canonical active 
            state of the multi-domain phosphatase SHP2.
  JOURNAL   bioRxiv [Preprint]. 2025 Jul 4:2025.07.02.662799. doi: 
            10.1101/2025.07.02.662799.
  PUBMED    40631229
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: "bioRxiv 
            [Preprint]. 2025 Jul 4:2025.07.02.662799. doi: 
            10.1101/2025.07.02.662799."
COMMENT     Sequence Label: pET-28b His6-TEV-SHP2-FL(R4A,R5A)
FEATURES             Location/Qualifiers
     source          1..7126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /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     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3485..3504
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3485..3503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3504..3528
                     /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             3543..3565
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3584..3601
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3623..3643
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5475..5492
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5545..5563)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5559..5606
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5643..6098
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5730..5749)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5940..5961
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6190..7005)
                     /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(6190..7005)
                     /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     6913..6932
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6997..7016
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     3481 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     3541 attttgttta actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca
     3601 cgattacgat atcccaacga ccgaaaacct ttacttccag ggccatatga catcggcggc
     3661 atggtttcac ccaaatatca ctggtgtgga ggcagaaaac ctactgttga caagaggagt
     3721 tgatggcagt tttttggcaa ggcctagtaa aagtaaccct ggagacttca cactttccgt
     3781 tagaagaaat ggagctgtca cccacatcaa gattcagaac actggtgatt actatgacct
     3841 gtatggaggg gagaaatttg ccactttggc tgagttggtc cagtattaca tggaacatca
     3901 cgggcaatta aaagagaaga atggagatgt cattgagctt aaatatcctc tgaactgtgc
     3961 agatcctacc tctgaaaggt ggtttcatgg acatctctct gggaaagaag cagagaaatt
     4021 attaactgaa aaaggaaaac atggtagttt tcttgtacga gagagccaga gccaccctgg
     4081 agattttgtt ctttctgtgc gcactggtga tgacaaaggg gagagcaatg acggcaagtc
     4141 taaagtgacc catgttatga ttcgctgtca ggaactgaaa tacgacgttg gtggaggaga
     4201 acggtttgat tctttgacag atcttgtgga acattataag aagaatccta tggtggaaac
     4261 attgggtaca gtactacaac tcaagcagcc ccttaacacg actcgtataa atgctgctga
     4321 aatagaaagc agagttcgag aactaagcaa attagctgag accacagata aagtcaaaca
     4381 aggcttttgg gaagaatttg agacactaca acaacaggag tgcaaacttc tctacagccg
     4441 aaaagagggt caaaggcaag aaaacaaaaa caaaaataga tataaaaaca tcctgccctt
     4501 tgatcatacc agggttgtcc tacacgatgg tgatcccaat gagcctgttt cagattacat
     4561 caatgcaaat atcatcatgc ctgaatttga aaccaagtgc aacaattcaa agcccaaaaa
     4621 gagttacatt gccacacaag gctgcctgca aaacacggtg aatgactttt ggcggatggt
     4681 gttccaagaa aactcccgag tgattgtcat gacaacgaaa gaagtggaga gaggaaagag
     4741 taaatgtgtc aaatactggc ctgatgagta tgctctaaaa gaatatggcg tcatgcgtgt
     4801 taggaacgtc aaagaaagcg ccgctcatga ctatacgcta agagaactta aactttcaaa
     4861 ggttggacaa gggaatacgg agagaacggt ctggcaatac cactttcgga cctggccgga
     4921 ccacggcgtg cccagcgacc ctgggggcgt gctggacttc ctggaggagg tgcaccataa
     4981 gcaggagagc atcatggatg cagggccggt cgtggtgcac tgcagtgctg gaattggccg
     5041 gacagggacg ttcattgtga ttgatattct tattgacatc atcagagaga aaggtgttga
     5101 ctgcgatatt gacgttccca aaaccatcca gatggtgcgg tctcagaggt cagggatggt
     5161 ccagacagaa gcacagtacc gatttatcta tatggcggtc cagcattata ttgaaacact
     5221 acagcgcagg attgaagaag agcagaaaag caagaggaaa gggcacgaat atacaaatat
     5281 taagtattct ctagcggacc agacgagtgg agatcagagc cctctcccgc cttgtactcc
     5341 aacgccaccc tgtgcagaaa tgagagaaga cagtgctaga gtctatgaaa acgtgggcct
     5401 gatgcaacag cagaaaagtt tcagataggg atccgaattc gagctccgtc gacaagcttg
     5461 cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     5521 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     5581 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     5641 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     5701 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     5761 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     5821 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     5881 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     5941 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     6001 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     6061 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     6121 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     6181 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     6241 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     6301 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     6361 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     6421 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     6481 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     6541 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     6601 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     6661 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     6721 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     6781 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     6841 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     6901 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     6961 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     7021 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     7081 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
