LOCUS       sequence-192265-        6909 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            ITSN1-3/5.
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_ITSN1-3/5
FEATURES             Location/Qualifiers
     source          1..6909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..32
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        55..132
                     /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             133..1215
                     /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(152..171)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1458..1463,1464..1479,1480..1486)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1494..1510
                     /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     1499..1521
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1595..1612
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1628..2281
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2239..2261
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2288..2311
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2324..2341
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2561..2608
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4201..4656)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4338..4359)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4550..4569
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4698..4720)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4806..4946
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4861..4880
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4961..4978)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5132..5719)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5890..6681,6682..6750))
                     /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     6513..6532
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6751..6855)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat tcccgacacc
       61 atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
      121 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
      181 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
      241 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
      301 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
      361 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
      421 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
      481 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
      541 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
      601 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
      661 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
      721 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
      781 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
      841 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
      901 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
      961 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     1021 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     1081 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     1141 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     1201 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacaatt
     1261 ctcatgtttg acagcttatc atcgactgca cggtgcacca atgcttctgg cgtcaggcag
     1321 ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc
     1381 aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa
     1441 tattctgaaa tgagctgttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag
     1501 cggataacaa tttcacacag gaaacagcca gtccgtttag gtgttttcac gagcacttca
     1561 ccaacaagga ccatagatta tgaaaatcga agaacaccat caccatcacc attccagcgg
     1621 taagcttatg tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg
     1681 acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg
     1741 tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta
     1801 tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa
     1861 gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc
     1921 ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct
     1981 caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg
     2041 tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc
     2101 tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg
     2161 ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta
     2221 tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa
     2281 atcggatctg gaagttctgt tccaggggcc cctgtccagc ggtctggttc cgcgtggttc
     2341 cggtaccgcg gcccagccgg ccatggccca gggcgcgctg gaagaattta tcgccatgta
     2401 cacctacgaa tcctccgaac aaggtgatct gacctttcag cagggtgatg ttattctggt
     2461 taccaaaaaa gatggtgatt ggtggaccgg tactgttggt gataaagccg gtgttttccc
     2521 atccaactat gtccgtctga aagatgcggc cgcataataa accgatacaa ttaaaggctc
     2581 cttttggagc cttttttttg gagattttca acgtgaaaaa attattattc gcaattcctt
     2641 tagttgttcc tttctattct cactccgctg aaactgttga aagttgttta gcaaaacccc
     2701 atacagaaaa ttcatttact aacgtctgga aagacgacaa aactttagat cgttacgcta
     2761 actatgaggg ctgtctgtgg aatgctacag gcgttgtagt ttgtactggt gacgaaactc
     2821 agtgtctagc tagagtggcg gtggctctgg ttccggtgat tttgattatg aaaagatggc
     2881 aaacgctaat aagggggcta tgaccgaaaa tgccgatgaa aacgcgctac agtctgacgc
     2941 taaaggcaaa cttgattctg tcgctactga ttacggtgct gctatcgatg gtttcattgg
     3001 tgacgtttcc ggccttgcta atggtaatgg tgctactggt gattttgctg gctctaattc
     3061 ccaaatggct caagtcggtg acggtgataa ttcaccttta atgaataatt tccgtcaata
     3121 tttaccttcc ctccctcaat cggttgaatg tcgccctttt gtctttggcg ctggtaaacc
     3181 atatgaattt tctattgatt gtgacaaaat aaacttattc cgtggtgtct ttgcgtttct
     3241 tttatatgtt gccaccttta tgtatgtatt ttctacgttt gctaacatac tgcgtaataa
     3301 ggagtcttaa tcatgccagt tcttttggct agcgccgccc tataccttgt ctgcctcccc
     3361 gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc
     3421 tcgctaacgg attcaccact ccaagaattg gagccaatca attcttgcgg agaactgtga
     3481 atgcgcaaac caacccttgg cagaacatat ccatcgcgtc cgccatctcc agcagccgca
     3541 cgcggcgcat ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt
     3601 cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga
     3661 tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat
     3721 gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca
     3781 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat
     3841 ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc
     3901 ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa
     3961 cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc
     4021 ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct
     4081 ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac
     4141 aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc aggatccgcg
     4201 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     4261 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     4321 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     4381 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     4441 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     4501 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     4561 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     4621 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcgcg gatcctgcct cgcgcgtttc
     4681 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     4741 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     4801 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg
     4861 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat
     4921 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     4981 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5041 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5101 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5161 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5221 agggtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5281 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5341 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5401 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5461 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5521 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     5581 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5641 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5701 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5761 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5821 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5881 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5941 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6001 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6061 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6121 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6181 tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat
     6241 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatattgtg
     6301 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6361 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6421 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6481 accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt
     6541 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6601 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6661 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6721 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6781 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6841 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     6901 tatcatgac
//
