LOCUS       sequence-192617-        6912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            SH3RF2-2/3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6912)
  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 6912)
  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"; 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_SH3RF2-2/3
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..44
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        67..144
                     /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             145..1227
                     /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(164..183)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1470..1475,1476..1491,1492..1498)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1506..1522
                     /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     1511..1533
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1607..1624
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1640..2293
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2251..2273
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2300..2323
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2336..2353
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2576..2623
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4216..4671)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4353..4374)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4565..4584
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4713..4735)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4821..4961
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4876..4895
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4976..4993)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5147..5734)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5905..6696,6697..6765))
                     /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     6528..6547
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6766..6870)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga
       61 attcccgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa
      121 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
      181 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
      241 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
      301 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
      361 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
      421 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
      481 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
      541 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
      601 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
      661 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
      721 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
      781 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
      841 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
      901 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
      961 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     1021 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     1081 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     1141 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     1201 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     1261 ttaggcacaa ttctcatgtt tgacagctta tcatcgactg cacggtgcac caatgcttct
     1321 ggcgtcaggc agccatcgga agctgtggta tggctgtgca ggtcgtaaat cactgcataa
     1381 ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac
     1441 ggttctggca aatattctga aatgagctgt tgacaattaa tcatcggctc gtataatgtg
     1501 tggaattgtg agcggataac aatttcacac aggaaacagc cagtccgttt aggtgttttc
     1561 acgagcactt caccaacaag gaccatagat tatgaaaatc gaagaacacc atcaccatca
     1621 ccattccagc ggtaagctta tgtcccctat actaggttat tggaaaatta agggccttgt
     1681 gcaacccact cgacttcttt tggaatatct tgaagaaaaa tatgaagagc atttgtatga
     1741 gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa
     1801 tcttccttat tatattgatg gtgatgttaa attaacacag tctatggcca tcatacgtta
     1861 tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat
     1921 gcttgaagga gcggttttgg atattagata cggtgtttcg agaattgcat atagtaaaga
     1981 ctttgaaact ctcaaagttg attttcttag caagctacct gaaatgctga aaatgttcga
     2041 agatcgttta tgtcataaaa catatttaaa tggtgatcat gtaacccatc ctgacttcat
     2101 gttgtatgac gctcttgatg ttgttttata catggaccca atgtgcctgg atgcgttccc
     2161 aaaattagtt tgttttaaaa aacgtattga agctatccca caaattgata agtacttgaa
     2221 atccagcaag tatatagcat ggcctttgca gggctggcaa gccacgtttg gtggtggcga
     2281 ccatcctcca aaatcggatc tggaagttct gttccagggg cccctgtcca gcggtctggt
     2341 tccgcgtggt tccggtaccg cggcccagcc ggccccactg tgccgtgcac tgtacaactt
     2401 tgatctgcgt ggtaaagaca aatccgaaaa ccaggattgt ctgacctttc tgaaagatga
     2461 cattatcacc gtcatttccc gtgttgatga aaactgggcc gaaggtaaac tgggtgataa
     2521 agttggtatc tttccgattc tgtttgtcga accgaacctg gcggccgcat aataaaccga
     2581 tacaattaaa ggctcctttt ggagcctttt ttttggagat tttcaacgtg aaaaaattat
     2641 tattcgcaat tcctttagtt gttcctttct attctcactc cgctgaaact gttgaaagtt
     2701 gtttagcaaa accccataca gaaaattcat ttactaacgt ctggaaagac gacaaaactt
     2761 tagatcgtta cgctaactat gagggctgtc tgtggaatgc tacaggcgtt gtagtttgta
     2821 ctggtgacga aactcagtgt ctagctagag tggcggtggc tctggttccg gtgattttga
     2881 ttatgaaaag atggcaaacg ctaataaggg ggctatgacc gaaaatgccg atgaaaacgc
     2941 gctacagtct gacgctaaag gcaaacttga ttctgtcgct actgattacg gtgctgctat
     3001 cgatggtttc attggtgacg tttccggcct tgctaatggt aatggtgcta ctggtgattt
     3061 tgctggctct aattcccaaa tggctcaagt cggtgacggt gataattcac ctttaatgaa
     3121 taatttccgt caatatttac cttccctccc tcaatcggtt gaatgtcgcc cttttgtctt
     3181 tggcgctggt aaaccatatg aattttctat tgattgtgac aaaataaact tattccgtgg
     3241 tgtctttgcg tttcttttat atgttgccac ctttatgtat gtattttcta cgtttgctaa
     3301 catactgcgt aataaggagt cttaatcatg ccagttcttt tggctagcgc cgccctatac
     3361 cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg
     3421 gaagccggcg gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct
     3481 tgcggagaac tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca
     3541 tctccagcag ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca
     3601 tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc
     3661 agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga
     3721 cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga
     3781 agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct
     3841 gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct
     3901 ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat
     3961 gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc
     4021 ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc
     4081 ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc
     4141 tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt
     4201 accgcaggat ccgcgaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     4261 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     4321 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     4381 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     4441 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     4501 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     4561 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     4621 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcgcggatcc
     4681 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     4741 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     4801 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
     4861 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     4921 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     4981 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5041 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5101 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5161 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5221 gactataaag ataccagggt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5281 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5341 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5401 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5461 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5521 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5581 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5641 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5701 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5761 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5821 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5881 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5941 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6001 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6061 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6121 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6181 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg
     6241 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6301 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6361 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6421 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6481 atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc
     6541 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6601 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6661 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6721 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6781 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6841 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6901 ctaagaaacc at
//
