LOCUS       sequence-191077-        6900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            SH3RF3-1/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6900)
  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 6900)
  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_SH3RF3-1/2
FEATURES             Location/Qualifiers
     source          1..6900
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..37
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        60..137
                     /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             138..1220
                     /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(157..176)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1463..1468,1469..1484,1485..1491)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1499..1515
                     /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     1504..1526
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1600..1617
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1633..2286
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2244..2266
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2293..2316
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2329..2346
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2557..2604
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4197..4652)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4334..4355)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4546..4565
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4694..4716)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4802..4942
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4857..4876
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4957..4974)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5128..5715)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5886..6677,6678..6746))
                     /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     6509..6528
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6747..6851)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattcccg
       61 acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
      121 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
      181 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
      241 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
      301 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
      361 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
      421 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
      481 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
      541 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
      601 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
      661 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
      721 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
      781 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
      841 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
      901 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
      961 ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
     1021 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
     1081 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
     1141 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     1201 gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca
     1261 caattctcat gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca
     1321 ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt
     1381 cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg
     1441 gcaaatattc tgaaatgagc tgttgacaat taatcatcgg ctcgtataat gtgtggaatt
     1501 gtgagcggat aacaatttca cacaggaaac agccagtccg tttaggtgtt ttcacgagca
     1561 cttcaccaac aaggaccata gattatgaaa atcgaagaac accatcacca tcaccattcc
     1621 agcggtaagc ttatgtcccc tatactaggt tattggaaaa ttaagggcct tgtgcaaccc
     1681 actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta tgagcgcgat
     1741 gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc caatcttcct
     1801 tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg ttatatagct
     1861 gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc aatgcttgaa
     1921 ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa agactttgaa
     1981 actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt
     2041 ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt catgttgtat
     2101 gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt cccaaaatta
     2161 gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt gaaatccagc
     2221 aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg cgaccatcct
     2281 ccaaaatcgg atctggaagt tctgttccag gggcccctgt ccagcggtct ggttccgcgt
     2341 ggttccggta ccgcggccca gccggccaat gtctacctgg cactgtacgc atataaaccg
     2401 caaaaatcag atgaactgga actgcataaa ggcgaaatgt atcgtgtcct ggaaaaatgc
     2461 caggatggct ggtttaaagg tgcgagcctg cgtaccggcg tgtctggtgt tttcccgggc
     2521 aactatgtca cgccggttag tgcggccgca taataaaccg atacaattaa aggctccttt
     2581 tggagccttt tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt
     2641 tgttcctttc tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aaccccatac
     2701 agaaaattca tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta
     2761 tgagggctgt ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg
     2821 tctagctaga gtggcggtgg ctctggttcc ggtgattttg attatgaaaa gatggcaaac
     2881 gctaataagg gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc tgacgctaaa
     2941 ggcaaacttg attctgtcgc tactgattac ggtgctgcta tcgatggttt cattggtgac
     3001 gtttccggcc ttgctaatgg taatggtgct actggtgatt ttgctggctc taattcccaa
     3061 atggctcaag tcggtgacgg tgataattca cctttaatga ataatttccg tcaatattta
     3121 ccttccctcc ctcaatcggt tgaatgtcgc ccttttgtct ttggcgctgg taaaccatat
     3181 gaattttcta ttgattgtga caaaataaac ttattccgtg gtgtctttgc gtttctttta
     3241 tatgttgcca cctttatgta tgtattttct acgtttgcta acatactgcg taataaggag
     3301 tcttaatcat gccagttctt ttggctagcg ccgccctata ccttgtctgc ctccccgcgt
     3361 tgcgtcgcgg tgcatggagc cgggccacct cgacctgaat ggaagccggc ggcacctcgc
     3421 taacggattc accactccaa gaattggagc caatcaattc ttgcggagaa ctgtgaatgc
     3481 gcaaaccaac ccttggcaga acatatccat cgcgtccgcc atctccagca gccgcacgcg
     3541 gcgcatctcg ggcagcgttg ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt
     3601 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg
     3661 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat
     3721 ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat
     3781 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt
     3841 attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac
     3901 cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg
     3961 tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc
     4021 ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat
     4081 cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc
     4141 agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagga tccgcgaaat
     4201 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     4261 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     4321 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     4381 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     4441 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     4501 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     4561 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     4621 cgccgcgctt aatgcgccgc tacagggcgc gtcgcggatc ctgcctcgcg cgtttcggtg
     4681 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     4741 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     4801 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
     4861 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     4921 aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4981 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5041 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5101 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5161 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggg
     5221 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5281 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5341 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5401 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5461 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5521 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     5581 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5641 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5701 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5761 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5821 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5881 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5941 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6001 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6061 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6121 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6181 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6241 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6301 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6361 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6421 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6481 agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa
     6541 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6601 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6661 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6721 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6781 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6841 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
//
