LOCUS       sequence-192251-        6909 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            ITSN1-2/5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6909)
  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 6909)
  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_ITSN1-2/5
FEATURES             Location/Qualifiers
     source          1..6909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     12..30
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        53..130
                     /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             131..1213
                     /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(150..169)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1456..1461,1462..1477,1478..1484)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1492..1508
                     /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     1497..1519
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1593..1610
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1626..2279
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2237..2259
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2286..2309
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2322..2339
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2559..2606
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4199..4654)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4336..4357)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4548..4567
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4696..4718)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4804..4944
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4859..4878
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4959..4976)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5130..5717)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5888..6679,6680..6748))
                     /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     6511..6530
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6749..6853)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattc ccgacaccat
       61 cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
      121 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
      181 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
      241 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
      301 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
      361 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
      421 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
      481 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
      541 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
      601 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
      661 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
      721 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
      781 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
      841 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
      901 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
      961 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     1021 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     1081 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     1141 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     1201 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcacaattct
     1261 catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc
     1321 atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa
     1381 ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata
     1441 ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga attgtgagcg
     1501 gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga gcacttcacc
     1561 aacaaggacc atagattatg aaaatcgaag aacaccatca ccatcaccat tccagcggta
     1621 agcttatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac
     1681 ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg
     1741 ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata
     1801 ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc
     1861 acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg
     1921 ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca
     1981 aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc
     2041 ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc
     2101 ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt
     2161 ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata
     2221 tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaat
     2281 cggatctgga agttctgttc caggggcccc tgtccagcgg tctggttccg cgtggttccg
     2341 gtaccgcggc ccagccggcc atggcccagg gcgcgctgct gcaagcacaa gcattgtatc
     2401 catggcgtgc caaaaaagat aaccatctga actttaacaa aaacgatgtc attaccgtcc
     2461 tggaacaaca agatatgtgg tggtttggtg aagttcaggg tcagaaaggc tggtttccga
     2521 aatcctatgt taaactgatt tccgcggccg cataataaac cgatacaatt aaaggctcct
     2581 tttggagcct tttttttgga gattttcaac gtgaaaaaat tattattcgc aattccttta
     2641 gttgttcctt tctattctca ctccgctgaa actgttgaaa gttgtttagc aaaaccccat
     2701 acagaaaatt catttactaa cgtctggaaa gacgacaaaa ctttagatcg ttacgctaac
     2761 tatgagggct gtctgtggaa tgctacaggc gttgtagttt gtactggtga cgaaactcag
     2821 tgtctagcta gagtggcggt ggctctggtt ccggtgattt tgattatgaa aagatggcaa
     2881 acgctaataa gggggctatg accgaaaatg ccgatgaaaa cgcgctacag tctgacgcta
     2941 aaggcaaact tgattctgtc gctactgatt acggtgctgc tatcgatggt ttcattggtg
     3001 acgtttccgg ccttgctaat ggtaatggtg ctactggtga ttttgctggc tctaattccc
     3061 aaatggctca agtcggtgac ggtgataatt cacctttaat gaataatttc cgtcaatatt
     3121 taccttccct ccctcaatcg gttgaatgtc gcccttttgt ctttggcgct ggtaaaccat
     3181 atgaattttc tattgattgt gacaaaataa acttattccg tggtgtcttt gcgtttcttt
     3241 tatatgttgc cacctttatg tatgtatttt ctacgtttgc taacatactg cgtaataagg
     3301 agtcttaatc atgccagttc ttttggctag cgccgcccta taccttgtct gcctccccgc
     3361 gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga atggaagccg gcggcacctc
     3421 gctaacggat tcaccactcc aagaattgga gccaatcaat tcttgcggag aactgtgaat
     3481 gcgcaaacca acccttggca gaacatatcc atcgcgtccg ccatctccag cagccgcacg
     3541 cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg
     3601 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
     3661 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
     3721 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
     3781 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
     3841 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
     3901 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
     3961 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
     4021 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
     4081 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
     4141 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag gatccgcgaa
     4201 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     4261 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     4321 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     4381 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     4441 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     4501 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     4561 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     4621 cccgccgcgc ttaatgcgcc gctacagggc gcgtcgcgga tcctgcctcg cgcgtttcgg
     4681 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     4741 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     4801 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg
     4861 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     4921 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4981 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5041 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5101 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5161 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5221 ggtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5281 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5341 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5401 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5461 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5521 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     5581 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5641 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5701 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5761 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5821 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5881 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5941 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6001 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6061 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6121 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6181 tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6241 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atattgtgca
     6301 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6361 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6421 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6481 cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa
     6541 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6601 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6661 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6721 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6781 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6841 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     6901 tcatgacat
//
