LOCUS       sequence-485202-        8367 bp    DNA     circular SYN 03-SEP-2025
DEFINITION  Expression vector with a clover-tagged phosphatase-inactive Shp2 
            mutant (D425A,C459S).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8367)
  AUTHORS   Conway JRW, Joshi O, Kaivola J, Follain G, Gounis M, Kuhl D, Ivaska 
            J
  TITLE     Dynamic regulation of integrin beta1 phosphorylation supports 
            invasion of breast cancer cells.
  JOURNAL   Nat Cell Biol. 2025 May 26. doi: 10.1038/s41556-025-01663-4.
  PUBMED    40419795
REFERENCE   2  (bases 1 to 8367)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 3, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Cell 
            Biol. 2025 May 26. doi: 10.1038/s41556-025-01663-4."
COMMENT     Sequence Label: pEF.DEST51-PTPN11(D425A,C459S)-Clover
FEATURES             Location/Qualifiers
     source          1..8367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(166..185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        473..1651
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          704..1642
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1599..1619
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1668..1687
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1668..1686
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1718..1742
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(3584..3586,3587..3589,3590..4300)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(3629..3650)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     4237..4258
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4315..4339)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4365..4406
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4416..4433
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4456..4473)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4462..4686
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4732..5160
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4819..4838)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5029..5050
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5169..5189)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5174..5503
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5354..5489
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5416..5435
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5551..5598
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5617..6015
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    6173..6294
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6210..6229)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6264..6283
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6343..6359)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6343..6359)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6356..6378)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6367..6383
                     /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)."
     promoter        complement(join(6391..6397,6398..6415,6416..6421))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6436..6457
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6574..6591)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6734..7322)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6814..6833)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7493..8284,8285..8353))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8116..8135
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8354..8367,1..91))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
       61 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
      121 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
      181 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      241 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      301 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      361 ttgattattg actaggcttt tgcaaaaagc tttgcaaaga tggataaagt tttaaacaga
      421 gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg
      481 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
      541 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
      601 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
      661 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg
      721 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac
      781 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc
      841 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg
      901 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa
      961 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag
     1021 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg
     1081 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc
     1141 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc
     1201 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg
     1261 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg
     1321 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc
     1381 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc
     1441 gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc
     1501 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt
     1561 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca
     1621 aagttttttt cttccatttc aggtgtcgtg aggaattagc ttggtactaa tacgactcac
     1681 tatagggaga cccaagctgg ctaggtaagc ttgatcaaca agtttgtaca aaaaagcagg
     1741 ctggcgccgg aaccaattca gtcgactgga tccggtaccg aggagatctg ccgccgcgat
     1801 cgccatgaca tcgcggagat ggtttcaccc aaatatcact ggtgtggagg cagaaaacct
     1861 actgttgaca agaggagttg atggcagttt tttggcaagg cctagtaaaa gtaaccctgg
     1921 agacttcaca ctttccgtta gaagaaatgg agctgtcacc cacatcaaga ttcagaacac
     1981 tggtgattac tatgacctgt atggagggga gaaatttgcc actttggctg agttggtcca
     2041 gtattacatg gaacatcacg ggcaattaaa agagaagaat ggagatgtca ttgagcttaa
     2101 atatcctctg aactgtgcag atcctacctc tgaaaggtgg tttcatggac atctctctgg
     2161 gaaagaagca gagaaattat taactgaaaa aggaaaacat ggtagttttc ttgtacgaga
     2221 gagccagagc caccctggag attttgttct ttctgtgcgc actggtgatg acaaagggga
     2281 gagcaatgac ggcaagtcta aagtgaccca tgttatgatt cgctgtcagg aactgaaata
     2341 cgacgttggt ggaggagaac ggtttgattc tttgacagat cttgtggaac attataagaa
     2401 gaatcctatg gtggaaacat tgggtacagt actacaactc aagcagcccc ttaacacgac
     2461 tcgtataaat gctgctgaaa tagaaagcag agttcgagaa ctaagcaaat tagctgagac
     2521 cacagataaa gtcaaacaag gcttttggga agaatttgag acactacaac aacaggagtg
     2581 caaacttctc tacagccgaa aagagggtca aaggcaagaa aacaaaaaca aaaatagata
     2641 taaaaacatc ctgccctttg atcataccag ggttgtccta cacgatggtg atcccaatga
     2701 gcctgtttca gattacatca atgcaaatat catcatgcct gaatttgaaa ccaagtgcaa
     2761 caattcaaag cccaaaaaga gttacattgc cacacaaggc tgcctgcaaa acacggtgaa
     2821 tgacttttgg cggatggtgt tccaagaaaa ctcccgagtg attgtcatga caacgaaaga
     2881 agtggagaga ggaaagagta aatgtgtcaa atactggcct gatgagtatg ctctaaaaga
     2941 atatggcgtc atgcgtgtta ggaacgtcaa agaaagcgcc gctcatgact atacgctaag
     3001 agaacttaaa ctttcaaagg ttggacaagg gaatacggag agaacggtct ggcaatacca
     3061 ctttcggacc tggccggccc acggcgtgcc cagcgaccct gggggcgtgc tggacttcct
     3121 ggaggaggtg caccataagc aggagagcat catggatgca gggccggtcg tggtgcacag
     3181 cagtgctgga attggccgga cagggacgtt cattgtgatt gatattctta ttgacatcat
     3241 cagagagaaa ggtgttgact gcgatattga cgttcccaaa accatccaga tggtgcggtc
     3301 tcagaggtca gggatggtcc agacagaagc acagtaccga tttatctata tggcggtcca
     3361 gcattatatt gaaacactac agcgcaggat tgaagaagag cagaaaagca agaggaaagg
     3421 gcacgaatat acaaatatta agtattctct agcggaccag acgagtggag atcagagccc
     3481 tctcccgcct tgtactccaa cgccaccctg tgcagaaatg agagaagaca gtgctagagt
     3541 ctatgaaaac gtgggcctga tgcaacagca gaaaagcggc cgcatggtga gcaagggcga
     3601 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     3661 caagttcagc gtccgcggcg agggcgaggg cgatgccacc aacggcaagc tgaccctgaa
     3721 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcgg
     3781 ctacggcgtg gcctgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     3841 gtccgccatg cccgaaggct acgtccagga gcgcaccatc tctttcaagg acgacggtac
     3901 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     3961 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaactt
     4021 caacagccac aacgtctata tcacggccga caagcagaag aacggcatca aggctaactt
     4081 caagatccgc cacaacgttg aggacggcag cgtgcagctc gccgaccact accagcagaa
     4141 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gccatcagtc
     4201 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     4261 cgccgccggg attacacatg gcatggacga gctgtacaag tagtaagaat ctagacccag
     4321 ctttcttgta caaagtggtt gatctagagg gcccgcggtt cgaaggtaag cctatcccta
     4381 accctctcct cggtctcgat tctacgcgta ccggtcatca tcaccatcac cattgagttt
     4441 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     4501 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     4561 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     4621 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     4681 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     4741 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     4801 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     4861 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4921 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     4981 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5041 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     5101 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5161 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     5221 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     5281 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     5341 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     5401 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     5461 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     5521 tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc
     5581 ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagccttt gtctcaagaa
     5641 gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac
     5701 tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta
     5761 tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg
     5821 gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc
     5881 ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg
     5941 aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat
     6001 gtgtgggagg gctaagcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt
     6061 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     6121 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     6181 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     6241 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     6301 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     6361 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     6421 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     6481 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     6541 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     6601 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     6661 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc caggaaccgt aaaaaggccg
     6721 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     6781 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     6841 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     6901 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6961 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     7021 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     7081 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     7141 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     7201 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     7261 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     7321 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     7381 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     7441 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     7501 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     7561 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     7621 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     7681 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     7741 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     7801 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     7861 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     7921 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7981 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     8041 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     8101 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     8161 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     8221 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     8281 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     8341 gttgaatact catactcttc ctttttc
//
