LOCUS       sequence-192236-        6939 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            RIMBP3-2/3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6939)
  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 6939)
  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_RIMBP3-2/3
FEATURES             Location/Qualifiers
     source          1..6939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..24
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        47..124
                     /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             125..1207
                     /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(144..163)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1450..1455,1456..1471,1472..1478)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1486..1502
                     /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     1491..1513
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1587..1604
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1620..2273
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2231..2253
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2280..2303
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2316..2333
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2583..2630
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4223..4678)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4360..4381)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4572..4591
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4720..4742)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4828..4968
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4883..4902
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4983..5000)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5154..5741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5912..6703,6704..6772))
                     /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     6535..6554
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6773..6877)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attcccgaca ccatcgaatg
       61 gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
      121 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
      181 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
      241 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
      301 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
      361 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
      421 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
      481 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
      541 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
      601 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
      661 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
      721 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
      781 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
      841 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
      901 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
      961 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     1021 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     1081 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     1141 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     1201 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcacaa ttctcatgtt
     1261 tgacagctta tcatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga
     1321 agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca
     1381 ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga
     1441 aatgagctgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac
     1501 aatttcacac aggaaacagc cagtccgttt aggtgttttc acgagcactt caccaacaag
     1561 gaccatagat tatgaaaatc gaagaacacc atcaccatca ccattccagc ggtaagctta
     1621 tgtcccctat actaggttat tggaaaatta agggccttgt gcaacccact cgacttcttt
     1681 tggaatatct tgaagaaaaa tatgaagagc atttgtatga gcgcgatgaa ggtgataaat
     1741 ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa tcttccttat tatattgatg
     1801 gtgatgttaa attaacacag tctatggcca tcatacgtta tatagctgac aagcacaaca
     1861 tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat gcttgaagga gcggttttgg
     1921 atattagata cggtgtttcg agaattgcat atagtaaaga ctttgaaact ctcaaagttg
     1981 attttcttag caagctacct gaaatgctga aaatgttcga agatcgttta tgtcataaaa
     2041 catatttaaa tggtgatcat gtaacccatc ctgacttcat gttgtatgac gctcttgatg
     2101 ttgttttata catggaccca atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa
     2161 aacgtattga agctatccca caaattgata agtacttgaa atccagcaag tatatagcat
     2221 ggcctttgca gggctggcaa gccacgtttg gtggtggcga ccatcctcca aaatcggatc
     2281 tggaagttct gttccagggg cccctgtcca gcggtctggt tccgcgtggt tccggtaccg
     2341 cggcccagcc ggccatggcc cagggcgcgc tgcgtgtttt tgttgccctg tccgattaca
     2401 atccactggt tatgtccgca gatctgaaag cagccgaaga agaactggtc tttcagaaac
     2461 gtcaactgct gcgtgtttgg ggttctcaag atacccatga tttctatctg tccgaatgca
     2521 accgtcaagt tggtaacatt ccgggtcgtc tggttgcaga aatggaagcg gccgcataat
     2581 aaaccgatac aattaaaggc tccttttgga gccttttttt tggagatttt caacgtgaaa
     2641 aaattattat tcgcaattcc tttagttgtt cctttctatt ctcactccgc tgaaactgtt
     2701 gaaagttgtt tagcaaaacc ccatacagaa aattcattta ctaacgtctg gaaagacgac
     2761 aaaactttag atcgttacgc taactatgag ggctgtctgt ggaatgctac aggcgttgta
     2821 gtttgtactg gtgacgaaac tcagtgtcta gctagagtgg cggtggctct ggttccggtg
     2881 attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg
     2941 aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg
     3001 ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg
     3061 gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt
     3121 taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt
     3181 ttgtctttgg cgctggtaaa ccatatgaat tttctattga ttgtgacaaa ataaacttat
     3241 tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta ttttctacgt
     3301 ttgctaacat actgcgtaat aaggagtctt aatcatgcca gttcttttgg ctagcgccgc
     3361 cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac
     3421 ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat tggagccaat
     3481 caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat atccatcgcg
     3541 tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc ctggccacgg
     3601 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact
     3661 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg
     3721 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
     3781 acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg
     3841 ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt
     3901 tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac
     3961 cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc
     4021 attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa
     4081 accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc
     4141 aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat
     4201 gagctttacc gcaggatccg cgaaattgta agcgttaata ttttgttaaa attcgcgtta
     4261 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     4321 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     4381 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     4441 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     4501 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     4561 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     4621 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg
     4681 cggatcctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     4741 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     4801 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     4861 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     4921 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
     4981 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5041 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5101 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5161 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5221 ccgacaggac tataaagata ccagggtttc cccctggaag ctccctcgtg cgctctcctg
     5281 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5341 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5401 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5461 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5521 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5581 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5641 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5701 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5761 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5821 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5881 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5941 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6001 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6061 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6121 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6181 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg
     6241 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6301 ttacatgatc ccccatattg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6361 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6421 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6481 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata
     6541 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6601 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6661 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6721 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6781 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6841 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6901 ctgacgtcta agaaaccatt attatcatga cattaacct
//
