LOCUS       sequence-192215-        6954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            TRIO-2/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6954)
  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 6954)
  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-2/2
FEATURES             Location/Qualifiers
     source          1..6954
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..43
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        66..143
                     /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             144..1226
                     /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(163..182)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1469..1474,1475..1490,1491..1497)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1505..1521
                     /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     1510..1532
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1606..1623
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1639..2292
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2250..2272
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2299..2322
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2335..2352
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2617..2664
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4257..4712)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4394..4415)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4606..4625
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4754..4776)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4862..5002
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4917..4936
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5017..5034)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5188..5775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5946..6737,6738..6806))
                     /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     6569..6588
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6807..6911)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa
       61 ttcccgacac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
      121 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
      181 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
      241 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
      301 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
      361 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
      421 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
      481 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
      541 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
      601 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
      661 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
      721 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
      781 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
      841 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
      901 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
      961 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     1021 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     1081 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     1141 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     1201 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     1261 taggcacaat tctcatgttt gacagcttat catcgactgc acggtgcacc aatgcttctg
     1321 gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat
     1381 tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg
     1441 gttctggcaa atattctgaa atgagctgtt gacaattaat catcggctcg tataatgtgt
     1501 ggaattgtga gcggataaca atttcacaca ggaaacagcc agtccgttta ggtgttttca
     1561 cgagcacttc accaacaagg accatagatt atgaaaatcg aagaacacca tcaccatcac
     1621 cattccagcg gtaagcttat gtcccctata ctaggttatt ggaaaattaa gggccttgtg
     1681 caacccactc gacttctttt ggaatatctt gaagaaaaat atgaagagca tttgtatgag
     1741 cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat
     1801 cttccttatt atattgatgg tgatgttaaa ttaacacagt ctatggccat catacgttat
     1861 atagctgaca agcacaacat gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg
     1921 cttgaaggag cggttttgga tattagatac ggtgtttcga gaattgcata tagtaaagac
     1981 tttgaaactc tcaaagttga ttttcttagc aagctacctg aaatgctgaa aatgttcgaa
     2041 gatcgtttat gtcataaaac atatttaaat ggtgatcatg taacccatcc tgacttcatg
     2101 ttgtatgacg ctcttgatgt tgttttatac atggacccaa tgtgcctgga tgcgttccca
     2161 aaattagttt gttttaaaaa acgtattgaa gctatcccac aaattgataa gtacttgaaa
     2221 tccagcaagt atatagcatg gcctttgcag ggctggcaag ccacgtttgg tggtggcgac
     2281 catcctccaa aatcggatct ggaagttctg ttccaggggc ccctgtccag cggtctggtt
     2341 ccgcgtggtt ccggtaccgc ggcccagccg gccatggccc agggcgcgct gtctaccatg
     2401 ctggtcaccc atgattacac cgccgttaaa gaagatgaaa tcaacgtcta tcagggtgaa
     2461 gttgttcaaa ttctggcctc caaccagcag aatatgtttc tggtttttcg tgcagccacc
     2521 gatcaatgtc cagcagcaga aggttggatt ccgggttttg ttctgggtca tacctccgcc
     2581 gttattgttg aaaacccgga tgcggccgca taataaaccg atacaattaa aggctccttt
     2641 tggagccttt tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt
     2701 tgttcctttc tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aaccccatac
     2761 agaaaattca tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta
     2821 tgagggctgt ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg
     2881 tctagctaga gtggcggtgg ctctggttcc ggtgattttg attatgaaaa gatggcaaac
     2941 gctaataagg gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc tgacgctaaa
     3001 ggcaaacttg attctgtcgc tactgattac ggtgctgcta tcgatggttt cattggtgac
     3061 gtttccggcc ttgctaatgg taatggtgct actggtgatt ttgctggctc taattcccaa
     3121 atggctcaag tcggtgacgg tgataattca cctttaatga ataatttccg tcaatattta
     3181 ccttccctcc ctcaatcggt tgaatgtcgc ccttttgtct ttggcgctgg taaaccatat
     3241 gaattttcta ttgattgtga caaaataaac ttattccgtg gtgtctttgc gtttctttta
     3301 tatgttgcca cctttatgta tgtattttct acgtttgcta acatactgcg taataaggag
     3361 tcttaatcat gccagttctt ttggctagcg ccgccctata ccttgtctgc ctccccgcgt
     3421 tgcgtcgcgg tgcatggagc cgggccacct cgacctgaat ggaagccggc ggcacctcgc
     3481 taacggattc accactccaa gaattggagc caatcaattc ttgcggagaa ctgtgaatgc
     3541 gcaaaccaac ccttggcaga acatatccat cgcgtccgcc atctccagca gccgcacgcg
     3601 gcgcatctcg ggcagcgttg ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt
     3661 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg
     3721 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat
     3781 ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat
     3841 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt
     3901 attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac
     3961 cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg
     4021 tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc
     4081 ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat
     4141 cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc
     4201 agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagga tccgcgaaat
     4261 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     4321 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     4381 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     4441 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     4501 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     4561 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     4621 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     4681 cgccgcgctt aatgcgccgc tacagggcgc gtcgcggatc ctgcctcgcg cgtttcggtg
     4741 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     4801 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     4861 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
     4921 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     4981 aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5041 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5101 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5161 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5221 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggg
     5281 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5341 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5401 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5461 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5521 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5581 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     5641 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5701 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5761 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5821 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5881 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5941 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6001 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6061 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6121 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6181 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6241 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6301 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat attgtgcaaa
     6361 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6421 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6481 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6541 agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa
     6601 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6661 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6721 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6781 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6841 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6901 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac catt
//
