LOCUS       sequence-353146-        6147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in bacterial cells. Tandem SH2 domains, M6-N269. 
            Can be used for crystallography..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6147)
  AUTHORS   Bashore FM, Katis VL, Du Y, Sikdar A, Wang D, Bradshaw WJ, Rygiel 
            KA, Leisner TM, Chalk R, Mishra S, Williams AC, Gileadi O, Brennan 
            PE, Wiley JC, Gockley J, Cary GA, Carter GW, Young JE, Pearce KH, Fu
            H, Axtman AD
  TITLE     Characterization of covalent inhibitors that disrupt the interaction
            between the tandem SH2 domains of SYK and FCER1G phospho-ITAM.
  JOURNAL   bioRxiv. 2023 Jul 29:2023.07.28.551026. doi: 
            10.1101/2023.07.28.551026. Preprint.
  PUBMED    37547005
REFERENCE   2  (bases 1 to 6147)
  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: "bioRxiv. 
            2023 Jul 29:2023.07.28.551026. doi: 10.1101/2023.07.28.551026. 
            Preprint."
COMMENT     Sequence Label: SYKA-c020
FEATURES             Location/Qualifiers
     source          1..6147
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             45..62
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             87..107
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             965..982
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1035..1053)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1049..1096
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1133..1588
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1220..1239)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1430..1451
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1680..2495)
                     /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             complement(1680..2495)
                     /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     2403..2422
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2487..2506
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2617..3205
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3106..3125
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3359..3376
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3391..3530
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3456..3475)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3616..3638
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3632..3823)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4632..5714)
                     /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     5676..5695
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5715..5792)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5998..6017
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6101..6120
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6101..6119
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6120..6144
                     /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)."
ORIGIN
        1 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaccat catcatcatc
       61 attcttctgg tgtagatctg ggtaccgaga acctgtactt ccaatccatg gctgacagcg
      121 ccaaccacct gcccttcttt ttcggcaaca tcacccggga ggaggcagaa gattacctgg
      181 tccagggggg catgagtgat gggctttatt tgctgcgcca gagccgcaac tacctgggtg
      241 gcttcgccct gtccgtggcc cacgggagga aggcacacca ctacaccatc gagcgggagc
      301 tgaatggcac ctacgccatc gccggtggca ggacccatgc cagccccgcc gacctctgcc
      361 actaccactc ccaggagtct gatggcctgg tctgcctcct caagaagccc ttcaaccggc
      421 cccaaggggt gcagcccaag actgggccct ttgaggattt gaaggaaaac ctcatcaggg
      481 aatatgtgaa gcagacatgg aacctgcagg gtcaggctct ggagcaggcc atcatcagtc
      541 agaagcctca gctggagaag ctgatcgcta ccacagccca tgaaaaaatg ccttggttcc
      601 atggaaaaat ctctcgggaa gaatctgagc aaattgtcct gataggatca aagacaaatg
      661 gaaagttcct gatccgagcc agagacaaca acggctccta cgccctgtgc ctgctgcacg
      721 aagggaaggt gctgcactat cgcatcgaca aagacaagac agggaagctc tccatccccg
      781 agggaaagaa gttcgacacg ctctggcagc tagtcgagca ttattcttat aaagcagatg
      841 gtttgttaag agttcttact gtcccatgtc aaaaaatcgg cacacaggga aatgttaatt
      901 gacagtaaag gtggatacgg atccgaattc gagctccgtc gacaagcttg cggccgcact
      961 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     1021 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1081 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     1141 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1201 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1261 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     1321 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     1381 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     1441 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     1501 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     1561 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     1621 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     1681 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     1741 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     1801 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     1861 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     1921 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     1981 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     2041 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     2101 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     2161 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     2221 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     2281 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     2341 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     2401 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     2461 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     2521 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2581 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2641 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2701 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2761 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2821 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2881 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2941 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3001 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3061 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3121 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3181 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3241 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3301 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3361 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3421 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3481 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     3541 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3601 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3661 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     3721 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     3781 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     3841 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     3901 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     3961 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     4021 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     4081 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     4141 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     4201 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     4261 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     4321 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     4381 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     4441 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     4501 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     4561 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     4621 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4681 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     4741 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     4801 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     4861 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     4921 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     4981 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     5041 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     5101 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5161 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5221 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5281 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     5341 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5401 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5461 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     5521 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     5581 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     5641 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     5701 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     5761 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     5821 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     5881 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     5941 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     6001 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     6061 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
     6121 gaattgtgag cggataacaa ttcccct
//
