LOCUS       sequence-191906-        6930 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            TRIO-1/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6930)
  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 6930)
  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_TRIO-1/2
FEATURES             Location/Qualifiers
     source          1..6930
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     33..51
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        74..151
                     /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             152..1234
                     /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(171..190)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1477..1482,1483..1498,1499..1505)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1513..1529
                     /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     1518..1540
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1614..1631
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1647..2300
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2258..2280
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2307..2330
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2343..2360
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2601..2648
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4241..4696)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4378..4399)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4590..4609
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4738..4760)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4846..4986
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4901..4920
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5001..5018)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5172..5759)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5930..6721,6722..6790))
                     /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     6553..6572
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6791..6895)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
       61 ttcaagaatt cccgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg
      121 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
      181 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
      241 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
      301 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
      361 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
      421 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
      481 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
      541 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
      601 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
      661 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
      721 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
      781 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
      841 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
      901 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
      961 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     1021 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     1081 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     1141 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     1201 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
     1261 tcactcatta ggcacaattc tcatgtttga cagcttatca tcgactgcac ggtgcaccaa
     1321 tgcttctggc gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac
     1381 tgcataattc gtgtcgctca aggcgcactc ccgttctgga taatgttttt tgcgccgaca
     1441 tcataacggt tctggcaaat attctgaaat gagctgttga caattaatca tcggctcgta
     1501 taatgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagccag tccgtttagg
     1561 tgttttcacg agcacttcac caacaaggac catagattat gaaaatcgaa gaacaccatc
     1621 accatcacca ttccagcggt aagcttatgt cccctatact aggttattgg aaaattaagg
     1681 gccttgtgca acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt
     1741 tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt
     1801 ttcccaatct tccttattat attgatggtg atgttaaatt aacacagtct atggccatca
     1861 tacgttatat agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga
     1921 tttcaatgct tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata
     1981 gtaaagactt tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa
     2041 tgttcgaaga tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg
     2101 acttcatgtt gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg
     2161 cgttcccaaa attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt
     2221 acttgaaatc cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg
     2281 gtggcgacca tcctccaaaa tcggatctgg aagttctgtt ccaggggccc ctgtccagcg
     2341 gtctggttcc gcgtggttcc ggtaccgcgg cccagccggc catggcccag ggcgcgctgg
     2401 aactgaccgt cgtcattcat gatttcaccg cctgcaattc caatgaactg accattcgtc
     2461 gtggtcaaac cgttgaagtt ctggaacgtc cgcatgataa accagattgg tgtctcgttc
     2521 gtaccactga tcgttctcca gcagcagaag gtttagttcc atgtggttcc ctgtgtattg
     2581 cccatgcggc cgcataataa accgatacaa ttaaaggctc cttttggagc cttttttttg
     2641 gagattttca acgtgaaaaa attattattc gcaattcctt tagttgttcc tttctattct
     2701 cactccgctg aaactgttga aagttgttta gcaaaacccc atacagaaaa ttcatttact
     2761 aacgtctgga aagacgacaa aactttagat cgttacgcta actatgaggg ctgtctgtgg
     2821 aatgctacag gcgttgtagt ttgtactggt gacgaaactc agtgtctagc tagagtggcg
     2881 gtggctctgg ttccggtgat tttgattatg aaaagatggc aaacgctaat aagggggcta
     2941 tgaccgaaaa tgccgatgaa aacgcgctac agtctgacgc taaaggcaaa cttgattctg
     3001 tcgctactga ttacggtgct gctatcgatg gtttcattgg tgacgtttcc ggccttgcta
     3061 atggtaatgg tgctactggt gattttgctg gctctaattc ccaaatggct caagtcggtg
     3121 acggtgataa ttcaccttta atgaataatt tccgtcaata tttaccttcc ctccctcaat
     3181 cggttgaatg tcgccctttt gtctttggcg ctggtaaacc atatgaattt tctattgatt
     3241 gtgacaaaat aaacttattc cgtggtgtct ttgcgtttct tttatatgtt gccaccttta
     3301 tgtatgtatt ttctacgttt gctaacatac tgcgtaataa ggagtcttaa tcatgccagt
     3361 tcttttggct agcgccgccc tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg
     3421 gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg attcaccact
     3481 ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac caacccttgg
     3541 cagaacatat ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat ctcgggcagc
     3601 gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc
     3661 tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc
     3721 gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt
     3781 gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc
     3841 atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg
     3901 cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc
     3961 tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc
     4021 tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca
     4081 gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta
     4141 acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg
     4201 cttcacgacc acgctgatga gctttaccgc aggatccgcg aaattgtaag cgttaatatt
     4261 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     4321 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     4381 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4441 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     4501 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     4561 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     4621 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     4681 ccgctacagg gcgcgtcgcg gatcctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     4741 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     4801 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     4861 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
     4921 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     4981 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5041 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5101 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5161 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5221 agtcagaggt ggcgaaaccc gacaggacta taaagatacc agggtttccc cctggaagct
     5281 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5341 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5401 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5461 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5521 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5581 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     5641 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5701 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5761 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5821 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5881 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5941 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6001 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6061 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6121 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6181 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6241 ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6301 cccaacgatc aaggcgagtt acatgatccc ccatattgtg caaaaaagcg gttagctcct
     6361 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6421 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6481 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6541 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6601 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6661 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6721 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6781 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6841 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6901 ttccccgaaa agtgccacct gacgtctaag
//
