LOCUS       sequence-422774-        5982 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SH2 domain of the Blk kinase with N-terminal
            Thrombin-cleavable 6xHis tag and SUMO and C-terminal AviTag for 
            Streptavidin bead functionalization for binder selections.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5982)
  AUTHORS   Gagoski D, Rube HT, Rastogi C, Melo LAN, Li X, Voleti R, Shah NH, 
            Bussemaker HJ
  TITLE     Accurate affinity models for SH2 domains from peptide binding assays
            and free-energy regression.
  JOURNAL   Protein Sci. 2025 Nov;34(11):e70317. doi: 10.1002/pro.70317.
  PUBMED    41088778
REFERENCE   2  (bases 1 to 5982)
  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: "Protein 
            Sci."; date: "2025-11"; volume: "34(11):e70317. doi"; pages: " 
            10.1002/pro.70317"
COMMENT     Sequence Label: pET28-His6-SUMO-Blk-SH2-AviTag
FEATURES             Location/Qualifiers
     source          1..5982
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      13..468
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(100..119)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     310..331
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(560..1375)
                     /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(560..1375)
                     /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     1283..1302
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1367..1386
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1497..2085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1986..2005
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2239..2256
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2271..2410
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2336..2355)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2496..2518
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2512..2703)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3512..4594)
                     /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     4556..4575
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4595..4672)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4878..4897
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4985..5004
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4985..5003
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5004..5028
                     /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)."
     RBS             5043..5065
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5085..5102
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5112..5129
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5136..5429
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             5751..5795
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5827..5844
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5897..5915)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5911..5958
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
       61 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      121 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      181 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
      241 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
      301 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
      361 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
      421 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
      601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
      661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
      721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
      781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
      841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
      901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
      961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     2341 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     2401 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     2461 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     2521 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     2581 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     2641 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     2701 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     2761 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     2821 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     2881 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     2941 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     3001 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     3061 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     3121 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     3181 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     3241 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     3301 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     3361 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     3421 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     3481 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3541 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3601 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     3661 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     3721 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     3781 ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     3841 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     3901 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     3961 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     4021 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     4081 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     4141 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     4201 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     4261 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     4321 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     4381 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     4441 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     4501 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     4561 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     4621 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     4681 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     4741 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     4801 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     4861 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     4921 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg
     4981 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     5041 attttgttta actttaagaa ggagatatac atatgggcag cagccatcat catcatcatc
     5101 acggcagcgg cctggtgccg cgcggcagcg ctagcatgtc ggactcagaa gtcaatcaag
     5161 aagctaagcc agaggtcaag ccagaagtca agcctgagac tcacatcaat ttaaaggtgt
     5221 ccgatggatc ttcagagatc ttcttcaaga tcaaaaagac cactccttta agaaggctga
     5281 tggaagcgtt cgctaaaaga cagggtaagg aaatggactc cttaagattc ttgtacgacg
     5341 gtattagaat tcaagctgat cagacccctg aagatttgga catggaggat aacgatatta
     5401 ttgaggctca cagagaacag attggtggtg tggagagcct ggaaatggaa aggtggttct
     5461 ttagatcaca gggtcggaag gaggctgaga ggcagcttct tgctccaatc aacaaggccg
     5521 gctcctttct tatcagagag agtgaaacca acaaaggtgc cttctccctg tctgtgaagg
     5581 atgtcaccac ccagggggag ctgatcaagc actataagat ccgctgcctg gatgaagggg
     5641 gctactacat ctccccccgg atcaccttcc cctcgctcca ggccctggtg cagcactatt
     5701 ctaagaaggg ggatggtcta tgccagaggc tgaccctgcc ctgtgtgcgc ggcctgaacg
     5761 atatttttga agcgcagaaa attgaatggc atgaataacg tcgacaagct tgcggccgca
     5821 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     5881 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     5941 gtcttgaggg gttttttgct gaaaggagga actatatccg ga
//
