LOCUS       sequence-192217-        6915 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            SH3RF3-2/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6915)
  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 6915)
  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_SH3RF3-2/2
FEATURES             Location/Qualifiers
     source          1..6915
                     /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      2560..2607
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4200..4655)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4337..4358)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4549..4568
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4697..4719)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4805..4945
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4860..4879
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4960..4977)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5131..5718)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5889..6680,6681..6749))
                     /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     6512..6531
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6750..6854)
                     /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 ctggaacgtt atcgtgtcgt tgtttcctat
     2401 ccaccacaat ccgaagccga aattgaactg aaagaaggtg atattgttct tgttcataaa
     2461 aaacgtgaag atggttggta caaaggtacg ctgcaacgta atggtcgtac cggtcttttt
     2521 cccggttcct ttgttgaatc cttcgcggcc gcataataaa ccgatacaat taaaggctcc
     2581 ttttggagcc ttttttttgg agattttcaa cgtgaaaaaa ttattattcg caattccttt
     2641 agttgttcct ttctattctc actccgctga aactgttgaa agttgtttag caaaacccca
     2701 tacagaaaat tcatttacta acgtctggaa agacgacaaa actttagatc gttacgctaa
     2761 ctatgagggc tgtctgtgga atgctacagg cgttgtagtt tgtactggtg acgaaactca
     2821 gtgtctagct agagtggcgg tggctctggt tccggtgatt ttgattatga aaagatggca
     2881 aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct
     2941 aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt
     3001 gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc
     3061 caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat
     3121 ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggcgc tggtaaacca
     3181 tatgaatttt ctattgattg tgacaaaata aacttattcc gtggtgtctt tgcgtttctt
     3241 ttatatgttg ccacctttat gtatgtattt tctacgtttg ctaacatact gcgtaataag
     3301 gagtcttaat catgccagtt cttttggcta gcgccgccct ataccttgtc tgcctccccg
     3361 cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct
     3421 cgctaacgga ttcaccactc caagaattgg agccaatcaa ttcttgcgga gaactgtgaa
     3481 tgcgcaaacc aacccttggc agaacatatc catcgcgtcc gccatctcca gcagccgcac
     3541 gcggcgcatc tcgggcagcg ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc
     3601 gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat
     3661 acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg
     3721 aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac
     3781 cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc
     3841 tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca
     3901 taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac
     3961 ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc
     4021 cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt
     4081 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca
     4141 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca ggatccgcga
     4201 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     4261 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     4321 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     4381 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     4441 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     4501 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     4561 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     4621 acccgccgcg cttaatgcgc cgctacaggg cgcgtcgcgg atcctgcctc gcgcgtttcg
     4681 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     4741 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     4801 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     4861 ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg
     4921 cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4981 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5041 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5101 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5161 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5221 gggtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5281 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5341 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5401 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5461 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5521 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     5581 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5641 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5701 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5761 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5821 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5881 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5941 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6001 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6061 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6121 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6181 ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg
     6241 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catattgtgc
     6301 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6361 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6421 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6481 ccgagttgct cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta
     6541 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6601 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6661 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6721 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6781 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6841 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     6901 atcatgacat taacc
//
