LOCUS       sequence-192375-        6933 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            VAV2-1/2*.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6933)
  AUTHORS   Teyra J, Huang H, Jain S, Guan X, Dong A, Liu Y, Tempel W, Min J, 
            Tong Y, Kim PM, Bader GD, Sidhu SS
  TITLE     Comprehensive Analysis of the Human SH3 Domain Family Reveals a Wide
            Variety of Non-canonical Specificities.
  JOURNAL   Structure. 2017 Oct 3;25(10):1598-1610.e3. doi: 
            10.1016/j.str.2017.07.017. Epub 2017 Sep 7.
  PUBMED    28890361
REFERENCE   2  (bases 1 to 6933)
  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"; doi: 
            "10.1016/j.str.2017.07.017"; journalName: "Structure"; date: 
            "2017-10-3- 3"; volume: "25"; issue: "10"; pages: "1598-1610.e3"
COMMENT     Sequence Label: pHH0103_VAV2-1/2*
FEATURES             Location/Qualifiers
     source          1..6933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..25
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        48..125
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             126..1208
                     /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(145..164)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1451..1456,1457..1472,1473..1479)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1487..1503
                     /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     1492..1514
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1588..1605
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1621..2274
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2232..2254
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2281..2304
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2317..2334
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2578..2625
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4218..4673)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4355..4376)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4567..4586
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4715..4737)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4823..4963
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4878..4897
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4978..4995)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5149..5736)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5907..6698,6699..6767))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6530..6549
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6768..6872)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tataaaaata ggcgtatcac gaggcccttt cgtcttcaag aattcccgac accatcgaat
       61 ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
      121 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
      181 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
      241 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
      301 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
      361 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
      421 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
      481 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
      541 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
      601 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
      661 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
      721 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
      781 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
      841 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
      901 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
      961 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     1021 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     1081 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     1141 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     1201 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcaca attctcatgt
     1261 ttgacagctt atcatcgact gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg
     1321 aagctgtggt atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc
     1381 actcccgttc tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg
     1441 aaatgagctg ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa
     1501 caatttcaca caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa
     1561 ggaccataga ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt
     1621 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt
     1681 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
     1741 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat
     1801 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac
     1861 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg
     1921 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt
     1981 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
     2041 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat
     2101 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa
     2161 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca
     2221 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat
     2281 ctggaagttc tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc
     2341 gcggcccagc cggccatggc ccagggcgcg ctgccgaaaa tggttgccgt ccagaactat
     2401 catggtaatc cagccccacc aggtaaacca gttctgacct tccaaaccgg tgatgttctg
     2461 gaactgctgc gtggtgatcc agaatctcca tggtgggaag gtcgtctggt tcaaacccgt
     2521 aaatccggtt actttccgtc ctcttccgtt aaaccatgtc cagcggccgc ataataaacc
     2581 gatacaatta aaggctcctt ttggagcctt ttttttggag attttcaacg tgaaaaaatt
     2641 attattcgca attcctttag ttgttccttt ctattctcac tccgctgaaa ctgttgaaag
     2701 ttgtttagca aaaccccata cagaaaattc atttactaac gtctggaaag acgacaaaac
     2761 tttagatcgt tacgctaact atgagggctg tctgtggaat gctacaggcg ttgtagtttg
     2821 tactggtgac gaaactcagt gtctagctag agtggcggtg gctctggttc cggtgatttt
     2881 gattatgaaa agatggcaaa cgctaataag ggggctatga ccgaaaatgc cgatgaaaac
     2941 gcgctacagt ctgacgctaa aggcaaactt gattctgtcg ctactgatta cggtgctgct
     3001 atcgatggtt tcattggtga cgtttccggc cttgctaatg gtaatggtgc tactggtgat
     3061 tttgctggct ctaattccca aatggctcaa gtcggtgacg gtgataattc acctttaatg
     3121 aataatttcc gtcaatattt accttccctc cctcaatcgg ttgaatgtcg cccttttgtc
     3181 tttggcgctg gtaaaccata tgaattttct attgattgtg acaaaataaa cttattccgt
     3241 ggtgtctttg cgtttctttt atatgttgcc acctttatgt atgtattttc tacgtttgct
     3301 aacatactgc gtaataagga gtcttaatca tgccagttct tttggctagc gccgccctat
     3361 accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa
     3421 tggaagccgg cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt
     3481 cttgcggaga actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc
     3541 catctccagc agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg
     3601 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta
     3661 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc
     3721 gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg
     3781 gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc
     3841 ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct
     3901 ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc
     3961 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
     4021 ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc
     4081 ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
     4141 gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
     4201 ttaccgcagg atccgcgaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     4261 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     4321 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     4381 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     4441 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     4501 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     4561 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     4621 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcgcggat
     4681 cctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     4741 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     4801 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     4861 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg
     4921 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     4981 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5041 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5101 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5161 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5221 aggactataa agataccagg gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5281 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5341 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5401 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5461 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5521 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5581 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5641 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5701 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5761 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5821 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5881 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5941 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6001 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6061 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6121 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6181 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca
     6241 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6301 tgatccccca tattgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6361 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6421 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6481 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg
     6541 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6601 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6661 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6721 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6781 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6841 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6901 gtctaagaaa ccattattat catgacatta acc
//
