LOCUS       sequence-192338-        6924 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            SH3RF1-2/4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6924)
  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 6924)
  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_SH3RF1-2/4
FEATURES             Location/Qualifiers
     source          1..6924
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..22
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        45..122
                     /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             123..1205
                     /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(142..161)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1448..1453,1454..1469,1470..1476)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1484..1500
                     /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     1489..1511
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1585..1602
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1618..2271
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2229..2251
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2278..2301
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2314..2331
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2566..2613
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4206..4661)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4343..4364)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4555..4574
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4703..4725)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4811..4951
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4866..4885
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4966..4983)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5137..5724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5895..6686,6687..6755))
                     /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     6518..6537
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6756..6860)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaaataggc gtatcacgag gccctttcgt cttcaagaat tcccgacacc atcgaatggt
       61 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
      121 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
      181 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
      241 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
      301 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
      361 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
      421 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
      481 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
      541 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
      601 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
      661 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
      721 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
      781 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
      841 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
      901 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
      961 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     1021 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     1081 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     1141 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     1201 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacaatt ctcatgtttg
     1261 acagcttatc atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag
     1321 ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact
     1381 cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa
     1441 tgagctgttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag cggataacaa
     1501 tttcacacag gaaacagcca gtccgtttag gtgttttcac gagcacttca ccaacaagga
     1561 ccatagatta tgaaaatcga agaacaccat caccatcacc attccagcgg taagcttatg
     1621 tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg
     1681 gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg
     1741 cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt
     1801 gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg
     1861 ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat
     1921 attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat
     1981 tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca
     2041 tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt
     2101 gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa
     2161 cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg
     2221 cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa atcggatctg
     2281 gaagttctgt tccaggggcc cctgtccagc ggtctggttc cgcgtggttc cggtaccgcg
     2341 gcccagccgg ccatggccca gggcgagctg ccacaatgca aagccctgta tgattttgaa
     2401 gttaaagata aagaagccga taaagattgt ctgccgttcg ccaaagatga tgtcctgacc
     2461 gttattcgtc gtgtcgatga aaactgggca gaaggtatgc tggcagacaa aatcggtatc
     2521 ttcccgattt cctacgtcga atttaactcc gcggccgcat aataaaccga tacaattaaa
     2581 ggctcctttt ggagcctttt ttttggagat tttcaacgtg aaaaaattat tattcgcaat
     2641 tcctttagtt gttcctttct attctcactc cgctgaaact gttgaaagtt gtttagcaaa
     2701 accccataca gaaaattcat ttactaacgt ctggaaagac gacaaaactt tagatcgtta
     2761 cgctaactat gagggctgtc tgtggaatgc tacaggcgtt gtagtttgta ctggtgacga
     2821 aactcagtgt ctagctagag tggcggtggc tctggttccg gtgattttga ttatgaaaag
     2881 atggcaaacg ctaataaggg ggctatgacc gaaaatgccg atgaaaacgc gctacagtct
     2941 gacgctaaag gcaaacttga ttctgtcgct actgattacg gtgctgctat cgatggtttc
     3001 attggtgacg tttccggcct tgctaatggt aatggtgcta ctggtgattt tgctggctct
     3061 aattcccaaa tggctcaagt cggtgacggt gataattcac ctttaatgaa taatttccgt
     3121 caatatttac cttccctccc tcaatcggtt gaatgtcgcc cttttgtctt tggcgctggt
     3181 aaaccatatg aattttctat tgattgtgac aaaataaact tattccgtgg tgtctttgcg
     3241 tttcttttat atgttgccac ctttatgtat gtattttcta cgtttgctaa catactgcgt
     3301 aataaggagt cttaatcatg ccagttcttt tggctagcgc cgccctatac cttgtctgcc
     3361 tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg gaagccggcg
     3421 gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct tgcggagaac
     3481 tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca tctccagcag
     3541 ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct
     3601 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
     3661 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
     3721 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc
     3781 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc
     3841 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg
     3901 catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc
     3961 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag
     4021 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc
     4081 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga
     4141 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcaggat
     4201 ccgcgaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     4261 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     4321 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     4381 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     4441 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     4501 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     4561 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     4621 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcgcggatcc tgcctcgcgc
     4681 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     4741 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     4801 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     4861 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     4921 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     4981 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5041 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5101 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5161 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5221 ataccagggt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5281 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5341 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5401 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5461 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5521 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     5581 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5641 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5701 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5761 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5821 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5881 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5941 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6001 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6061 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6121 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6181 aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt
     6241 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccata
     6301 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6361 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6421 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6481 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga
     6541 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6601 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6661 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6721 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6781 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6841 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     6901 attattatca tgacattaac ctat
//
