LOCUS       sequence-492628-        7109 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Src pH insensitive mutant  in Mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7109)
  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 7109)
  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: Flag-Src C185A H201A H492A E524A
FEATURES             Location/Qualifiers
     source          1..7109
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     463..482
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     716..733
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    850..871
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(886..891,892..909,910..916)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    924..940
                     /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     929..951
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     948..964
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     948..964
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1013..1134
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1024..1043)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1078..1097
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1310..2104)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1421..1440)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2031..2050
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2171..2500)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2185..2320
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2239..2258)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2485..2505
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2514..2942)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2624..2645)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2836..2855
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2988..3212
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3201..3218
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3241..3258)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3268..3309)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(3335..3358)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(3362..3385)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    3396..3420
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(5047..5071)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(5100..5119)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5101..5119)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5164..5367)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5193..5213)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5368..5747
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5919..5938
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6013..6117
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6118..6186,6187..6978)
                     /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     complement(6336..6355)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
       61 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
      121 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
      181 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
      241 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
      301 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
      361 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
      421 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
      481 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      541 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      601 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      661 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      721 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
      781 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
      841 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
      901 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
      961 tgaccatgat tacgccaagc tctagctaga ggtcgacggt atacagacat gataagatac
     1021 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     1081 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg
     1141 ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa
     1201 aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg
     1261 caggttgggc gtcgcttggt cggtcatttc gcgaacccca gagtcccgct cagaagaact
     1321 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     1381 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     1441 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc
     1501 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct
     1561 cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     1621 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
     1681 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
     1741 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
     1801 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
     1861 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt
     1921 cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
     1981 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat
     2041 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa
     2101 tcatgcgaaa cgatcctcat cctgtctctt gatcagatcc gaaaatggat atacaagctc
     2161 ccgggagctt tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata
     2221 gctcagaggc agaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg
     2281 gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga
     2341 ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     2401 gggactttcc acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
     2461 ctggggagcc tggggacttt ccacacccta actgacacac attccacaga attaattcgc
     2521 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2581 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2641 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2701 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2761 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2821 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2881 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2941 gtggggatac cccctagagc cccagctggt tctttccgcc tcagaagcca tagagcccac
     3001 cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt cctgccccac
     3061 cccacccccc agaatagaat gacacctact cagacaatgc gatgcaattt cctcatttta
     3121 ttaggaaagg acagtgggag tggcaccttc cagggtcaag gaaggcacgg gggaggggca
     3181 aacaacagat ggctggcaac tagaaggcac agtcgaggct gatcagcggg tttaaactca
     3241 atggtgatgg tgatgatgac cggtacgcgt agaatcgaga ccgaggagag ggttagggat
     3301 aggcttacct tcgaaccgcg ggccctctag atcacttatc gtcgtcatcc ttgtaatctc
     3361 ccttatcgtc gtcatccttg taatcctcta gatcaaccac tttgtacaag aaagctgggt
     3421 cgaggttctc cccgggctgg tactggggcg cggtggacgt gaagtagtcc tccaggaagg
     3481 cctgcaggta ctcgaaggtg ggccgctcct caggctcctt ccgccagcac tggcacatga
     3541 ggtcggccag ggactcggga cactccggcg ggcagggcat ccggtagccc cgctccacct
     3601 ggtccagcac ctcgcggttc accatcccag ggtagggcac ccgtcccttt gtggtgagct
     3661 cagtcagcag gatcccgaag gaccacacgt ccgacttgat ggtgaagcgg ccatagaggg
     3721 cagcttctgg agccgtccac ttgatgggga atttggcacc ttgccgcgcc gtgtactcat
     3781 tgtcttcaat gagccgagcc agcccaaagt cggccacttt gcacaccagg ttctctccca
     3841 ccaggatgtt ggctgcacga aggtcccggt ggacgtagtt catccgctcc acgtacgcca
     3901 tgcctgaggc gatctgagca gccatgtcca ccagctgagg cagccgcagg tacttgcctg
     3961 tctccccctt gagaaagtcc agcaaactcc ccttgctcat gtactccgtg acgatgtaaa
     4021 tgggctcctc tgaaaccaca gcatacaact gcaccagctt ctcatgcctc agcttcttca
     4081 tgacctgggc ctcctgcagg aaggcctctg gagacatcgt gccaggcttc agggttttga
     4141 tggccaccct ggtggtaccg ttccaggtcc ccatccacac ctcgccaaag cagccctggc
     4201 ccagcttgac ctccagccgc agcgactccc gagggatctc ccaggcatcc ttggccaggc
     4261 cctgagtctg cggcttggac gtggggcaca cggtggtgag gcggtggcac aggccatcgg
     4321 cgtgtttgga gtagtaggcc accagctgct gcaggctgtt gaactgggtg cgggaggtga
     4381 tgtagaagcc gccgctgtcc agcttgcgga tcttgtaggc cttcacgttg aggcccttgg
     4441 cgttgtcgaa gtcagacact gagagggcgt aggcaccttt cgtggtctca ctttctcgca
     4501 cgaggaaggt ccctctcggg ttctctgcat tgagcagtaa ccgctctgac tcccgtctgg
     4561 tgatcttgcc aaaataccac tcctcagcct ggatggagtc ggagggcgcc acgtagttgc
     4621 tggggatgta gcctgtctgt cctgtgctga gcgagtgggc cagccaccag tctccctctg
     4681 tgttgttgac aatctggagc cgctcgcctt tcttgaagga caggtctgtc tccgtcctag
     4741 actcatagtc atagagggcc acaaaggtgg tcactccacc ggccagcggg cccgccctct
     4801 gcggggaggt gacggtgtcc gaggagttga agcctccgaa cagcttgggc tcggcggccg
     4861 cgggggcgaa ggccgcgctg gggccgcggt ggccgtcggc cgaggctggc ttgctggggg
     4921 tctgcgaggc ggggaaagcg cccccgccag cgccgtgcac gttctcggcg ggctccagga
     4981 tgcggcgccg ctggctggca tccttgggct tgctcttgtt gctacccatg gttctatctc
     5041 cttcgaagcc tgcttttttg tacaaacttg ttgatagctt aactagccag cttgggtctc
     5101 cctatagtga gtcgtattaa tttcgataag ccagtaagca gtgggttctc tagttagcca
     5161 gagagctctg cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg
     5221 ggcggagttg ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc
     5281 attgacgtca atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc
     5341 attgatgtac tgccaaaacc gcatcaccat ggtaatagcg atgactaata cgtagatgta
     5401 ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt
     5461 accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa
     5521 gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt
     5581 tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc
     5641 aggcgggcca tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa
     5701 tgaccccgta attgattact attaataact agtcaataat caatgtcaac gcgtatatct
     5761 ggcccgtaca tcgcgaagca gcgcaaaacg cctaacccta agcagattct tcatgcaatt
     5821 gtcggtcaag ccttgccttg ttgtagctta aattttgctc gcgcactact cagcgacctc
     5881 caacacacaa gcagggagca gatactggct taactatgcg gcatcagagc agattgtact
     5941 gagagtgcac cataggggat cgggagatct cccgatccgt cgacgtcagg tggcactttt
     6001 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     6061 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     6121 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     6181 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     6241 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     6301 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     6361 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     6421 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     6481 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     6541 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6601 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6661 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6721 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6781 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6841 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6901 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6961 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     7021 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     7081 aaaatccctt aacgtgagtt ttcgttcca
//
