LOCUS       sequence-491601-        6944 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of pH insensitive SHP2 mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6944)
  AUTHORS   Van Dyck PK, Piszkin L, Gorski EA, Nascimento ET, Abebe JA, Hoffmann
            LM, Peng JW, White KA
  TITLE     Ionizable networks mediate pH-dependent allostery in the SH2 
            domain-containing signaling proteins SHP2 and SRC.
  JOURNAL   Sci Signal. 2025 Nov 11;18(912):eadt3018. doi: 
            10.1126/scisignal.adt3018. Epub 2025 Nov 11.
  PUBMED    41218103
REFERENCE   2  (bases 1 to 6944)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci 
            Signal."; date: "2025-11-11"; volume: "18(912):eadt3018. doi"; 
            pages: " 10.1126/scisignal.adt3018. Epub 2025 Nov 11"
COMMENT     Sequence Label: pGEX-4T1 GST-SHP2 H116A E252A
FEATURES             Location/Qualifiers
     source          1..6944
                     /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"
     promoter        833..910
                     /gene="lacI"
                     /label=lacI promoter
     CDS             911..1993
                     /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
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAMEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(930..949)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(2042..2047,2048..2065,2066..2072)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2080..2096
                     /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     2104..2120
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2104..2120
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2116..2643
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(2136..2153)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2136..2152)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2145..2167)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(2745..2750,2751..2766,2767..2773)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2781..2797
                     /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)."
     CDS             2820..3473
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     3431..3453
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             3480..3497
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(5556..5578)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5723..5741)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5809..5913
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5914..5982,5983..6774)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6132..6151)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, 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 cgcataaatt ccgacaccat
      841 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
      901 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
      961 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     1021 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     1081 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     1141 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     1201 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     1261 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctatgga
     1321 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     1381 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     1441 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     1501 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     1561 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     1621 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     1681 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     1741 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     1801 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     1861 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     1921 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     1981 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccccagg
     2041 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     2101 acacaggaaa cagctatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt
     2161 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2221 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2281 aatggcgaat ggcgctttgc ctggtttccg gcaccagaag cggtgccgga aagctggctg
     2341 gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct caaactggca gatgcacggt
     2401 tacgatgcgc ccatctacac caacgtgacc tatcccatta cggtcaatcc gccgtttgtt
     2461 cccacggaga atccgacggg ttgttactcg ctcacattta atgttgatga aagctggcta
     2521 caggaaggcc agacgcgaat tatttttgat ggcgttggaa ttagcttatc gactgcacgg
     2581 tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg
     2641 taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg
     2701 cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca attaatcatc
     2761 ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagtattca
     2821 tgtcccctat actaggttat tggaaaatta agggccttgt gcaacccact cgacttcttt
     2881 tggaatatct tgaagaaaaa tatgaagagc atttgtatga gcgcgatgaa ggtgataaat
     2941 ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa tcttccttat tatattgatg
     3001 gtgatgttaa attaacacag tctatggcca tcatacgtta tatagctgac aagcacaaca
     3061 tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat gcttgaagga gcggttttgg
     3121 atattagata cggtgtttcg agaattgcat atagtaaaga ctttgaaact ctcaaagttg
     3181 attttcttag caagctacct gaaatgctga aaatgttcga agatcgttta tgtcataaaa
     3241 catatttaaa tggtgatcat gtaacccatc ctgacttcat gttgtatgac gctcttgatg
     3301 ttgttttata catggaccca atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa
     3361 aacgtattga agctatccca caaattgata agtacttgaa atccagcaag tatatagcat
     3421 ggcctttgca gggctggcaa gccacgtttg gtggtggcga ccatcctcca aaatcggatc
     3481 tggttccgcg tggatcccca ggaattccgg gcgggaggaa catgacatcg cggagatggt
     3541 ttcacccaaa tatcactggt gtggaggcag aaaacctact gttgacaaga ggagttgatg
     3601 gcagtttttt ggcaaggcct agtaaaagta accctggaga cttcacactt tccgttagaa
     3661 gaaatggagc tgtcacccac atcaagattc agaacactgg tgattactat gacctgtatg
     3721 gaggggagaa atttgccact ttggctgagt tggtccagta ttacatggaa catcacgggc
     3781 aattaaaaga gaagaatgga gatgtcattg agcttaaata tcctctgaac tgtgcagatc
     3841 ctacctctga aaggtggttt catggagctc tctctgggaa agaagcagag aaattattaa
     3901 ctgaaaaagg aaaacatggt agttttcttg tacgagagag ccagagccac cctggagatt
     3961 ttgttctttc tgtgcgcact ggtgatgaca aaggggagag caatgacggc aagtctaaag
     4021 tgacccatgt tatgattcgc tgtcaggaac tgaaatacga cgttggtgga ggagaacggt
     4081 ttgattcttt gacagatctt gtggaacatt ataagaagaa tcctatggtg gaaacattgg
     4141 gtacagtact acaactcaag cagcccctta acacgactcg tataaatgct gctgaaatag
     4201 aaagcagagt tcgagaacta agcaaattag ctgagaccac agataaagtc aaacaaggct
     4261 tttgggaaga atttgcgaca ctacaacaac aggagtgcaa acttctctac agccgaaaag
     4321 agggtcaaag gcaagaaaac aaaaacaaaa atagatataa aaacatcctg ccctttgatc
     4381 ataccagggt tgtcctacac gatggtgatc ccaatgagcc tgtttcagat tacatcaatg
     4441 caaatatcat catgcctgaa tttgaaacca agtgcaacaa ttcaaagccc aaaaagagtt
     4501 acattgccac acaaggctgc ctgcaaaaca cggtgaatga cttttggcgg atggtgttcc
     4561 aagaaaactc ccgagtgatt gtcatgacaa cgaaagaagt ggagagagga aagagtaaat
     4621 gtgtcaaata ctggcctgat gagtatgctc taaaagaata tggcgtcatg cgtgttagga
     4681 acgtcaaaga aagcgccgct catgactata cgctaagaga acttaaactt tcaaaggttg
     4741 gacaagggaa tacggagaga acggtctggc aataccactt tcggacctgg ccggaccacg
     4801 gcgtgcccag cgaccctggg ggcgtgctgg acttcctgga ggaggtgcac cataagcagg
     4861 agagcatcat ggatgcaggg ccggtcgtgg tgcactgcag tgctggaatt ggccggacag
     4921 ggacgttcat tgtgattgat attcttattg acatcatcag agagaaaggt gttgactgcg
     4981 atattgacgt tcccaaaacc atccagatgg tgcggtctca gaggtcaggg atggtccaga
     5041 cagaagcaca gtaccgattt atctatatgg cggtccagca ttatattgaa acactacagc
     5101 gcaggattga agaagagcag aaaagcaaga ggaaagggca cgaatataca aatattaagt
     5161 attctctagc ggaccagacg agtggagatc agagccctct cccgccttgt actccaacgc
     5221 caccctgtgc agaaatgaga gaagacagtg ctagagtcta tgaaaacgtg ggcctgatgc
     5281 aacagcagaa aagtttcaga tgagaaaacc tgccaaaact tcagcacaga aatagatgtg
     5341 gactttcacc ctctccctaa aaagatcaag aacagacgca agaaagttta tgtgaagaca
     5401 gaatttggat ttggaaggct tgcaatgtgg ttgactacct tttgataagc aaaatttgaa
     5461 accatttaaa gaccggaatt cccgggtcga ctcgagcggc cgcatcgtga ctgactgacg
     5521 atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     5581 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     5641 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     5701 tataattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     5761 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5821 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5881 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5941 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6001 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6061 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6121 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     6181 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6241 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6301 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6361 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6421 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     6481 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6541 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6601 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6661 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6721 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6781 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6841 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6901 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc
//
