LOCUS       sequence-422834-        6060 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression of SH2 domain of the Grb2 with N-terminal 
            TEV-cleavable 6xHis tag, C-terminal mCherry, 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 6060)
  TITLE     bacterial expression plasmids for production of recombinant SH2 
            domains from the Src-family kinases
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6060)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pET28-His6-TEV-Grb2-SH2-mCherry-AviTag
FEATURES             Location/Qualifiers
     source          1..6060
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(218..1033)
                     /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(218..1033)
                     /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     941..960
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1025..1044
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1155..1743
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1644..1663
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1897..1914
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1929..2068
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1994..2013)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2154..2176
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2170..2361)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3170..4252)
                     /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     4214..4233
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4253..4330)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4536..4555
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4639..4658
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4639..4657
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4658..4682
                     /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             4697..4719
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4730..4747
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4754..5407
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             5423..5443
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             5941..5958
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6011..6029)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
       61 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
      121 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
      181 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
      241 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
      301 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
      361 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
      421 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
      481 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
      541 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
      601 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
      661 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
      721 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
      781 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
      841 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
      901 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
      961 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     1021 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     1081 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     1141 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     1201 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     1261 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     1321 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     1381 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     1441 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     1501 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     1561 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     1621 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     1681 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     1741 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     1801 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     1861 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     1921 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     1981 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     2041 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     2101 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     2161 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     2221 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     2281 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     2341 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     2401 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     2461 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     2521 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     2581 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     2641 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     2701 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     2761 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     2821 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     2881 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     2941 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     3001 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     3061 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     3121 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     3181 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3241 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     3301 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     3361 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     3421 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     3481 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     3541 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     3601 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     3661 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     3721 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     3781 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     3841 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     3901 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     3961 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     4021 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     4081 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     4141 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     4201 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     4261 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     4321 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     4381 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     4441 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     4501 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     4561 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     4621 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     4681 cccctctaga aataattttg tttaacttta agaaggagat atacatatgc accatcacca
     4741 ccatcactca tcgatgtctc ccattttggg ttattggaag ataaaggggt tagtacaacc
     4801 gaccagatta ctgttagagt accttgaaga gaagtatgaa gagcatttat atgagcgcga
     4861 cgaaggcgat aagtggcgca acaagaaatt tgagttaggc ctcgagtttc cgaaccttcc
     4921 ttattatatc gacggggacg taaagttgac gcaatctatg gcaataattc ggtatatagc
     4981 agacaagcat aacatgttgg gtggctgccc caaagagcgt gccgagatct ccatgttaga
     5041 aggagccgtc ctggacattc gctatggtgt gtcgcgcatt gcctactcaa aagatttcga
     5101 gactttaaaa gttgactttc tgagcaagct gccggagatg cttaagatgt tcgaagatcg
     5161 gctgtgtcac aagacgtacc tcaacggtga ccacgttacc cacccagact ttatgcttta
     5221 cgatgcgctg gacgttgtgc tttacatgga cccgatgtgt ctggacgcgt ttccgaagtt
     5281 ggtatgtttc aagaagcgga ttgaggctat tccccaaata gacaaatacc ttaagagtag
     5341 taagtacatc gcgtggccac ttcaaggttg gcaagctacc ttcggcggcg gcgaccatcc
     5401 cccaaagtcg ggcggcggca gcgagaacct gtacttccaa tccatgggaa agttcgggca
     5461 acaatcgggt gccatatatg tgggtaacta tcgtgtggtt aatcgccacc tcgctactca
     5521 caatgattgg gccaacttag tgtgggagga cagctctcgc gatctccttg tatcatcgac
     5581 cactgcgcag ggttgcgaca ccattgcgcg ctgtgattgc cagacaggcg tgtattattg
     5641 ctcgtctcgt cgtaaacatt atccggtttc tttttctaag ccgtcactga tctttgttga
     5701 agcgtcagag tactatccag cccgttatca gtcccatctg atgctggctg tgggtcacag
     5761 cgaacccggg gattgtggcg gtatactgcg ctgtcagcac ggtgtagttg gcattgtcag
     5821 tacaggaggt aatgggttag tcggatttgc cgatgtgcgg gatttactgt ggttggacga
     5881 ggaggctatg gaacagtaat gactcgagca cccgtcgaca agcttgcggc cgcactcgag
     5941 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     6001 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
//
