LOCUS       sequence-192255-        6921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            RASA1-1/1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6921)
  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 6921)
  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_RASA1-1/1
FEATURES             Location/Qualifiers
     source          1..6921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..38
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        61..138
                     /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             139..1221
                     /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(158..177)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1464..1469,1470..1485,1486..1492)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1500..1516
                     /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     1505..1527
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1601..1618
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1634..2287
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2245..2267
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2294..2317
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2330..2347
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2579..2626
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4219..4674)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4356..4377)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4568..4587
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4716..4738)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4824..4964
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4879..4898
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4979..4996)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5150..5737)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5908..6699,6700..6768))
                     /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     6531..6550
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6769..6873)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc aagaattccc
       61 gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
      121 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
      181 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
      241 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
      301 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
      361 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
      421 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
      481 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
      541 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
      601 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
      661 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
      721 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
      781 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
      841 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
      901 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
      961 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     1021 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     1081 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     1141 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     1201 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     1261 acaattctca tgtttgacag cttatcatcg actgcacggt gcaccaatgc ttctggcgtc
     1321 aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg
     1381 tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct
     1441 ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat
     1501 tgtgagcgga taacaatttc acacaggaaa cagccagtcc gtttaggtgt tttcacgagc
     1561 acttcaccaa caaggaccat agattatgaa aatcgaagaa caccatcacc atcaccattc
     1621 cagcggtaag cttatgtccc ctatactagg ttattggaaa attaagggcc ttgtgcaacc
     1681 cactcgactt cttttggaat atcttgaaga aaaatatgaa gagcatttgt atgagcgcga
     1741 tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc
     1801 ttattatatt gatggtgatg ttaaattaac acagtctatg gccatcatac gttatatagc
     1861 tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt gcagagattt caatgcttga
     1921 aggagcggtt ttggatatta gatacggtgt ttcgagaatt gcatatagta aagactttga
     1981 aactctcaaa gttgattttc ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg
     2041 tttatgtcat aaaacatatt taaatggtga tcatgtaacc catcctgact tcatgttgta
     2101 tgacgctctt gatgttgttt tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt
     2161 agtttgtttt aaaaaacgta ttgaagctat cccacaaatt gataagtact tgaaatccag
     2221 caagtatata gcatggcctt tgcagggctg gcaagccacg tttggtggtg gcgaccatcc
     2281 tccaaaatcg gatctggaag ttctgttcca ggggcccctg tccagcggtc tggttccgcg
     2341 tggttccggt accgcggccc agccggccat ggcccagggc gcgctgcgtc gtgttcgtgc
     2401 aattctgccg tataccaaag ttccggatac cgatgaaatc agcttcctga aaggtgatat
     2461 gttcattgtc cataacgaac tggaagatgg ttggatgtgg gttaccaatc tgcgtaccga
     2521 tgaacagggt ctgattgttg aagatttggt cgaagaagtc ggtgcggccg cataataaac
     2581 cgatacaatt aaaggctcct tttggagcct tttttttgga gattttcaac gtgaaaaaat
     2641 tattattcgc aattccttta gttgttcctt tctattctca ctccgctgaa actgttgaaa
     2701 gttgtttagc aaaaccccat acagaaaatt catttactaa cgtctggaaa gacgacaaaa
     2761 ctttagatcg ttacgctaac tatgagggct gtctgtggaa tgctacaggc gttgtagttt
     2821 gtactggtga cgaaactcag tgtctagcta gagtggcggt ggctctggtt ccggtgattt
     2881 tgattatgaa aagatggcaa acgctaataa gggggctatg accgaaaatg ccgatgaaaa
     2941 cgcgctacag tctgacgcta aaggcaaact tgattctgtc gctactgatt acggtgctgc
     3001 tatcgatggt ttcattggtg acgtttccgg ccttgctaat ggtaatggtg ctactggtga
     3061 ttttgctggc tctaattccc aaatggctca agtcggtgac ggtgataatt cacctttaat
     3121 gaataatttc cgtcaatatt taccttccct ccctcaatcg gttgaatgtc gcccttttgt
     3181 ctttggcgct ggtaaaccat atgaattttc tattgattgt gacaaaataa acttattccg
     3241 tggtgtcttt gcgtttcttt tatatgttgc cacctttatg tatgtatttt ctacgtttgc
     3301 taacatactg cgtaataagg agtcttaatc atgccagttc ttttggctag cgccgcccta
     3361 taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga
     3421 atggaagccg gcggcacctc gctaacggat tcaccactcc aagaattgga gccaatcaat
     3481 tcttgcggag aactgtgaat gcgcaaacca acccttggca gaacatatcc atcgcgtccg
     3541 ccatctccag cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc
     3601 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt
     3661 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg
     3721 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc
     3781 ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac
     3841 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc
     3901 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg
     3961 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta
     4021 cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg
     4081 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg
     4141 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc
     4201 tttaccgcag gatccgcgaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     4261 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     4321 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     4381 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     4441 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     4501 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     4561 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     4621 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcgcgga
     4681 tcctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     4741 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     4801 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     4861 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt
     4921 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     4981 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5041 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5101 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5161 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5221 caggactata aagataccag ggtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5281 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5341 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5401 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5461 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5521 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5581 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5641 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5701 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5761 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5821 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5881 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5941 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6001 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6061 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6121 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6181 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc
     6241 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6301 atgatccccc atattgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6361 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6421 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6481 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc
     6541 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6601 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6661 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6721 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6781 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6841 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6901 cgtctaagaa accattatta t
//
