LOCUS       sequence-422793-        4513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the kinase domain of cSrc with a 
            TEV-cleavable N-terminal 6xHis affinity tag; For in vitro tyrosine 
            phosphorylation in peptides/proteins and for kinase specificity 
            screenings.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4513)
  AUTHORS   Shah NH, Lobel M, Weiss A, Kuriyan J
  TITLE     Fine-tuning of substrate preferences of the Src-family kinase Lck 
            revealed through a high-throughput specificity screen.
  JOURNAL   Elife. 2018 Mar 16;7. pii: 35190. doi: 10.7554/eLife.35190.
  PUBMED    29547119
REFERENCE   2  (bases 1 to 4513)
  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: "Elife."; 
            date: "2018-03-16"; volume: "7. pii: 35190. doi"; pages: " 
            10.7554/eLife.35190"
COMMENT     Sequence Label: pET23a-H6-TEV-cSrc
FEATURES             Location/Qualifiers
     source          1..4513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(798..817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     958..980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(974..1165)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1674..1693
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1674..1692
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1724..1746
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1765..1782
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1804..1824
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2687..2704
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2757..2775)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2771..2818
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2855..3310
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2942..2961)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3152..3173
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3336..3440
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3441..3509,3510..4301)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3659..3678)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4472..4513,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      841 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      901 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      961 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     1021 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     1081 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     1141 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta atggggattt ctgttcatgg
     1201 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     1261 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     1321 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     1381 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     1441 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     1501 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     1561 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     1621 tgcgcacccg tggccaggac ccaacgctgc ccgagatctc gatcccgcga aattaatacg
     1681 actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga
     1741 aggagatata ccatgggcag cagccatcat catcatcatc acgattacga tatcccaacg
     1801 accgaaaacc tttacttcca gggccatatg aaggatgcct gggagatccc tcgggagtcg
     1861 ctgcggctgg aggtcaagct gggccagggc tgctttggcg aggtgtggat ggggacctgg
     1921 aacggtacca ccagggtggc catcaaaacc ctgaagcctg gcacgatgtc tccagaggcc
     1981 ttcctgcagg aggcccaggt catgaagaag ctgaggcatg agaagctggt gcagttgtat
     2041 gctgtggttt cagaggagcc catttacatc gtcacggagt acatgagcaa ggggagtttg
     2101 ctggactttc tcaaggggga gacaggcaag tacctgcggc tgcctcagct ggtggacatg
     2161 gctgctcaga tcgcctcagg catggcgtac gtggagcgga tgaactacgt ccaccgggac
     2221 cttcgtgcag ccaacatcct ggtgggagag aacctggtgt gcaaagtggc cgactttggg
     2281 ctggctcggc tcattgaaga caatgagtac acggcgcggc aaggtgccaa attccccatc
     2341 aagtggacgg ctccagaagc tgccctctat ggccgcttca ccatcaagtc ggacgtgtgg
     2401 tccttcggga tcctgctgac tgagctcacc acaaagggac gggtgcccta ccctgggatg
     2461 gtgaaccgcg aggtgctgga ccaggtggag cggggctacc ggatgccctg cccgccggag
     2521 tgtcccgagt ccctgcacga cctcatgtgc cagtgctggc ggaaggagcc tgaggagcgg
     2581 cccaccttcg agtacctgca ggccttcctg gaggactact tcacgtccac cgagccctag
     2641 ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca
     2701 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     2761 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     2821 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2881 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2941 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3001 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3061 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3121 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3181 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3241 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3301 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3361 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3421 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3481 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3541 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3601 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     3661 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     3721 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     3781 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3841 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3901 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3961 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     4021 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4081 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4141 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4201 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4261 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4321 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4381 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4441 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4501 gctgcttgca aac
//
