LOCUS       sequence-191785-        6978 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            ITSN1-1/5-IS2/3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6978)
  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 6978)
  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_ITSN1-1/5-IS2/3
FEATURES             Location/Qualifiers
     source          1..6978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..31
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        54..131
                     /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             132..1214
                     /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(151..170)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1457..1462,1463..1478,1479..1485)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1493..1509
                     /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     1498..1520
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1594..1611
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1627..2280
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2238..2260
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2287..2310
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2323..2340
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2629..2676
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4269..4724)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4406..4427)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4618..4637
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4766..4788)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4874..5014
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4929..4948
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5029..5046)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5200..5787)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5958..6749,6750..6818))
                     /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     6581..6600
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6819..6923)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt cccgacacca
       61 tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
      121 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
      181 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
      241 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
      301 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
      361 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
      421 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
      481 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
      541 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
      601 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
      661 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
      721 gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg
      781 aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca
      841 tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca
      901 ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg
      961 aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg
     1021 ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc
     1081 agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg
     1141 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     1201 aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcacaattc
     1261 tcatgtttga cagcttatca tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc
     1321 catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca
     1381 aggcgcactc ccgttctgga taatgttttt tgcgccgaca tcataacggt tctggcaaat
     1441 attctgaaat gagctgttga caattaatca tcggctcgta taatgtgtgg aattgtgagc
     1501 ggataacaat ttcacacagg aaacagccag tccgtttagg tgttttcacg agcacttcac
     1561 caacaaggac catagattat gaaaatcgaa gaacaccatc accatcacca ttccagcggt
     1621 aagcttatgt cccctatact aggttattgg aaaattaagg gccttgtgca acccactcga
     1681 cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt
     1741 gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct tccttattat
     1801 attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat agctgacaag
     1861 cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg
     1921 gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt tgaaactctc
     1981 aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt
     2041 cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt gtatgacgct
     2101 cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa attagtttgt
     2161 tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc cagcaagtat
     2221 atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa
     2281 tcggatctgg aagttctgtt ccaggggccc ctgtccagcg gtctggttcc gcgtggttcc
     2341 ggtaccgcgg cccagccggc cggtccgctg acgatctctg ctcaagaaaa tgtgaaagtt
     2401 gtctattacc gtgccctgta tccgtttgaa tcccgtagcc atgatgaaat taccatccag
     2461 ccgggcgata ttgtgatggt tatcggttgc tgggttgacg aaagccaaac cggtgaaccg
     2521 ggttggctgg gcggtgaact gaaaggcaaa acgggttggt ttccggcgaa ctatgccgaa
     2581 aaaattccgg aaaatgaagt cccggcgccg gtggcggccg cataataaac cgatacaatt
     2641 aaaggctcct tttggagcct tttttttgga gattttcaac gtgaaaaaat tattattcgc
     2701 aattccttta gttgttcctt tctattctca ctccgctgaa actgttgaaa gttgtttagc
     2761 aaaaccccat acagaaaatt catttactaa cgtctggaaa gacgacaaaa ctttagatcg
     2821 ttacgctaac tatgagggct gtctgtggaa tgctacaggc gttgtagttt gtactggtga
     2881 cgaaactcag tgtctagcta gagtggcggt ggctctggtt ccggtgattt tgattatgaa
     2941 aagatggcaa acgctaataa gggggctatg accgaaaatg ccgatgaaaa cgcgctacag
     3001 tctgacgcta aaggcaaact tgattctgtc gctactgatt acggtgctgc tatcgatggt
     3061 ttcattggtg acgtttccgg ccttgctaat ggtaatggtg ctactggtga ttttgctggc
     3121 tctaattccc aaatggctca agtcggtgac ggtgataatt cacctttaat gaataatttc
     3181 cgtcaatatt taccttccct ccctcaatcg gttgaatgtc gcccttttgt ctttggcgct
     3241 ggtaaaccat atgaattttc tattgattgt gacaaaataa acttattccg tggtgtcttt
     3301 gcgtttcttt tatatgttgc cacctttatg tatgtatttt ctacgtttgc taacatactg
     3361 cgtaataagg agtcttaatc atgccagttc ttttggctag cgccgcccta taccttgtct
     3421 gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga atggaagccg
     3481 gcggcacctc gctaacggat tcaccactcc aagaattgga gccaatcaat tcttgcggag
     3541 aactgtgaat gcgcaaacca acccttggca gaacatatcc atcgcgtccg ccatctccag
     3601 cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt
     3661 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga
     3721 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc
     3781 aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
     3841 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac
     3901 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg
     3961 ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc
     4021 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa
     4081 cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat
     4141 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
     4201 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
     4261 gatccgcgaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     4321 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     4381 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     4441 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     4501 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     4561 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     4621 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     4681 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcgcgga tcctgcctcg
     4741 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     4801 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     4861 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     4921 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     4981 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
     5041 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5101 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5161 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5221 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5281 aagataccag ggtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5341 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5401 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5461 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5521 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5581 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     5641 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5701 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5761 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5821 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5881 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5941 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6001 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6061 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6121 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6181 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6241 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
     6301 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6361 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6421 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6481 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6541 atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc
     6601 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6661 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6721 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6781 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6841 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6901 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     6961 accattatta tcatgaca
//
