LOCUS       sequence-487860-        8561 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Proximity-labeling proteomics with SHP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8561)
  AUTHORS   van Vlimmeren AE, Tang LC, Jiang Z, Iyer A, Voleti R, Krismer K, 
            Gaublomme JT, Jovanovic M, Shah NH
  TITLE     Proximity-labeling proteomics reveals remodeled interactomes and 
            altered localization of pathogenic SHP2 variants.
  JOURNAL   bioRxiv [Preprint]. 2025 Mar 21:2025.02.26.640373. doi: 
            10.1101/2025.02.26.640373.
  PUBMED    40661614
REFERENCE   2  (bases 1 to 8561)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 Mar 21:2025.02.26.640373. doi: 
            10.1101/2025.02.26.640373."
COMMENT     Sequence Label: pEF myc-SHP2(T42A)-TurboID
FEATURES             Location/Qualifiers
     source          1..8561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        135..182
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             201..599
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    757..878
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(794..813)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     848..867
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(927..943)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(927..943)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(940..962)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    951..967
                     /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(975..981,982..999,1000..1005))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1020..1041
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1158..1175)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1318..1906)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1398..1417)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2077..2868,2869..2937))
                     /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"
     promoter        complement(2938..3042)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3117..3136)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3424..4602
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3655..4593
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4550..4570
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     4619..4638
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4619..4637
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4759..4788
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6586..7542
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG
                     ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI
                     TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK
                     EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             7549..7575
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(7601..7618)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7607..7831
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7877..8305
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7964..7983)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8174..8195
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8314..8334)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(8319..8561,1..87)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(8499..8561,1..73)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     join(8561,1..19)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
       61 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
      121 tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
      181 acaaggtgag gaactaaacc atggccaagc ctttgtctca agaagaatcc accctcattg
      241 aaagagcaac ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg
      301 cagctctctc tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg
      361 gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga
      421 cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc
      481 gacaggtgct tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac
      541 agccgacggc agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag
      601 cacttcgtgg ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc
      661 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
      721 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
      781 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
      841 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
      901 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
      961 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     1021 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     1081 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1141 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     1201 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     1261 ggaaagaaca tgtgagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     1321 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     1381 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     1441 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     1501 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     1561 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     1621 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     1681 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     1741 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     1801 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     1861 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     1921 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     1981 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     2041 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     2101 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     2161 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     2221 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     2281 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     2341 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     2401 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     2461 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     2521 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     2581 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     2641 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     2701 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     2761 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     2821 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     2881 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     2941 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     3001 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     3061 gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca
     3121 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     3181 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
     3241 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
     3301 acgcgttgac attgattatt gactaggctt ttgcaaaaag ctttgcaaag atggataaag
     3361 ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa aggagtggga
     3421 attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
     3481 gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa
     3541 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
     3601 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt
     3661 gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga
     3721 attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
     3781 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
     3841 tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
     3901 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc
     3961 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
     4021 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
     4081 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
     4141 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
     4201 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
     4261 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
     4321 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
     4381 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     4441 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     4501 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     4561 acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggaattag cttggtacta
     4621 atacgactca ctatagggag acccaagctg gctaggtaag cttggtaccg agctcggatc
     4681 cactagtcca gtgtggtgga attctgcaga tatccagcac agtggcggcc gctcgagtct
     4741 agagggccct tcaccatgga acaaaaactc atctcagaag aggatctggg agggcagtct
     4801 gggcagatga catcgcggag atggtttcac ccaaatatca ctggtgtgga ggcagaaaac
     4861 ctactgttga caagaggagt tgatggcagt tttttggcaa ggcctagtaa aagtaaccct
     4921 ggagacttcg cactttccgt tagaagaaat ggagctgtca cccacatcaa gattcagaac
     4981 actggtgatt actatgacct gtatggaggg gagaaatttg ccactttggc tgagttggtc
     5041 cagtattaca tggaacatca cgggcaatta aaagagaaga atggagatgt cattgagctt
     5101 aaatatcctc tgaactgtgc agatcctacc tctgaaaggt ggtttcatgg acatctctct
     5161 gggaaagaag cagagaaatt attaactgaa aaaggaaaac atggtagttt tcttgtacga
     5221 gagagccaga gccaccctgg agattttgtt ctttctgtgc gcactggtga tgacaaaggg
     5281 gagagcaatg acggcaagtc taaagtgacc catgttatga ttcgctgtca ggaactgaaa
     5341 tacgacgttg gtggaggaga acggtttgat tctttgacag atcttgtgga acattataag
     5401 aagaatccta tggtggaaac attgggtaca gtactacaac tcaagcagcc ccttaacacg
     5461 actcgtataa atgctgctga aatagaaagc agagttcgag aactaagcaa attagctgag
     5521 accacagata aagtcaaaca aggcttttgg gaagaatttg agacactaca acaacaggag
     5581 tgcaaacttc tctacagccg aaaagagggt caaaggcaag aaaacaaaaa caaaaataga
     5641 tataaaaaca tcctgccctt tgatcatacc agggttgtcc tacacgatgg tgatcccaat
     5701 gagcctgttt cagattacat caatgcaaat atcatcatgc ctgaatttga aaccaagtgc
     5761 aacaattcaa agcccaaaaa gagttacatt gccacacaag gctgcctgca aaacacggtg
     5821 aatgactttt ggcggatggt gttccaagaa aactcccgag tgattgtcat gacaacgaaa
     5881 gaagtggaga gaggaaagag taaatgtgtc aaatactggc ctgatgagta tgctctaaaa
     5941 gaatatggcg tcatgcgtgt taggaacgtc aaagaaagcg ccgctcatga ctatacgcta
     6001 agagaactta aactttcaaa ggttggacaa gggaatacgg agagaacggt ctggcaatac
     6061 cactttcgga cctggccgga ccacggcgtg cccagcgacc ctgggggcgt gctggacttc
     6121 ctggaggagg tgcaccataa gcaggagagc atcatggatg cagggccggt cgtggtgcac
     6181 tgcagtgctg gaattggccg gacagggacg ttcattgtga ttgatattct tattgacatc
     6241 atcagagaga aaggtgttga ctgcgatatt gacgttccca aaaccatcca gatggtgcgg
     6301 tctcagaggt cagggatggt ccagacagaa gcacagtacc gatttatcta tatggcggtc
     6361 cagcattata ttgaaacact acagcgcagg attgaagaag agcagaaaag caagaggaaa
     6421 gggcacgaat atacaaatat taagtattct ctagcggacc agacgagtgg agatcagagc
     6481 cctctcccgc cttgtactcc aacgccaccc tgtgcagaaa tgagagaaga cagtgctaga
     6541 gtctatgaaa acgtgggcct gatgcaacag cagaaaagtt tcagaaaaga caatactgtg
     6601 cctctgaagc tgatcgctct cctggctaat ggcgagttcc atagtggcga acagctggga
     6661 gaaaccctgg gcatgtccag ggccgctatc aacaagcaca ttcagactct gcgcgactgg
     6721 ggcgtggacg tgttcaccgt gcccggaaag ggctactctc tgcccgagcc tatcccgctg
     6781 ctgaacgcta aacagattct gggacagctg gacggcggga gcgtggcagt cctgcctgtg
     6841 gtcgactcca ccaatcagta cctgctggat cgaatcggcg agctgaagag tggggatgct
     6901 tgcattgcag aatatcagca ggcagggaga ggaagcagag ggaggaaatg gttctctcct
     6961 tttggagcta acctgtacct gagtatgttt tggcgcctga agcggggacc agcagcaatc
     7021 ggcctgggcc cggtcatcgg aattgtcatg gcagaagcgc tgcgaaagct gggagcagac
     7081 aaggtgcgag tcaaatggcc caatgacctg tatctgcagg atagaaagct ggcaggcatc
     7141 ctggtggagc tggccggaat aacaggcgat gctgcacaga tcgtcattgg cgccgggatt
     7201 aacgtggcta tgaggcgcgt ggaggaaagc gtggtcaatc agggctggat cacactgcag
     7261 gaagcaggga ttaacctgga caggaatact ctggccgcta cgctgatccg agagctgcgg
     7321 gcagccctgg aactgttcga gcaggaaggc ctggctccat atctgccacg gtgggagaag
     7381 ctggataact tcatcaatag acccgtgaag ctgatcattg gggacaaaga gattttcggg
     7441 attagccggg ggattgataa acagggagcc ctgctgctgg aacaggacgg agttatcaaa
     7501 ccctggatgg gcggagaaat cagtctgcgg tctgccgaaa agctgcagct gcctcccctg
     7561 gagcgcctga ccctggacta agtttaaacc cgctgatcag cctcgactgt gccttctagt
     7621 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     7681 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     7741 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     7801 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     7861 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     7921 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     7981 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     8041 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     8101 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     8161 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     8221 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     8281 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     8341 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     8401 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     8461 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     8521 gttccgccca ttctccgccc catggctgac taattttttt t
//
