LOCUS       sequence-215717-        5549 bp    DNA     circular SYN 15-APR-2025
DEFINITION  express murine Syk (C)SH2 domain (His 162 to Gln 264), in E.coli 
            strain Rosetta 2 (DE3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5549)
  AUTHORS   Feng C, Roy A, Post CB
  TITLE     Entropic allostery dominates the phosphorylation-dependent 
            regulation of Syk tyrosine kinase release from immunoreceptor 
            tyrosine-based activation motifs.
  JOURNAL   Protein Sci. 2018 Jul 27. doi: 10.1002/pro.3489.
  PUBMED    30051939
REFERENCE   2  (bases 1 to 5549)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protein 
            Sci. 2018 Jul 27. doi: 10.1002/pro.3489."
COMMENT     Sequence Label: pET-30a(+)-C-SH2
FEATURES             Location/Qualifiers
     source          1..5549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             486..508
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    523..547
                     /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     complement(547..566)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(548..566)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(654..673)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        879..956
                     /gene="lacI"
                     /label=lacI promoter
     CDS             957..2039
                     /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     complement(976..995)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2848..3039
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3033..3055)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3141..3280
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3196..3215
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3295..3312)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3466..4054)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3546..3565)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4165..4184)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4176..4991
                     /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             4176..4991
                     /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     complement(4249..4268)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5083..5538)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5220..5241)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5432..5451
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagttattgt gcaccgatct
      181 tctggcatgg taccgtgagg actcttagta gcccatctgg cttgtaagag taatgttcca
      241 ctagctgcca gagggtgtca aacttcttcc cctcaggaat ggagagcttc ccggtcttgt
      301 ccctgtcaat gcggtagtgc aatactttcc cttcgtgcag caggcacaga gcataggagc
      361 cgctgttgtc tctggccctg atcaggaatt ttccattggt ctttgaccct atgaggaccg
      421 tctgctctga ttcatctctg gagatgttgc catggaacca gggcatcttc tcatgcatat
      481 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
      541 caattcccct atagtgagtc gtattaattt cgcgggatcg agatcgatct cgatcctcta
      601 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
      661 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt
      721 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca
      781 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
      841 aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa
      901 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
      961 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     1021 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     1081 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     1141 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     1201 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     1261 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     1321 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     1381 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     1441 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     1501 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     1561 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     1621 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     1681 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     1741 tgcgcgttgg tgcggacatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     1801 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     1861 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     1921 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     1981 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     2041 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc
     2101 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc
     2161 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg
     2221 gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg
     2281 gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt
     2341 ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg
     2401 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     2461 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca
     2521 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg
     2581 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     2641 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
     2701 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
     2761 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
     2821 agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg
     2881 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg
     2941 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc
     3001 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     3061 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     3121 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
     3181 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat tgcggtgtga
     3241 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct
     3301 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3361 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3421 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3481 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3541 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3601 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3661 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3721 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3781 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3841 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3901 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3961 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4021 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4081 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac
     4141 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
     4201 cttgctctag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     4261 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg
     4321 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     4381 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     4441 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg
     4501 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     4561 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     4621 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     4681 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
     4741 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     4801 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     4861 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     4921 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     4981 agtttttcta agaattaatt catgagcgga tacatatttg aatgtattta gaaaaataaa
     5041 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     5101 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5161 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5221 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5281 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5341 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5401 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5461 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5521 cgccgctaca gggcgcgtcc cattcgcca
//
