LOCUS       sequence-192391-        6918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            ITSN2-1/5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6918)
  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 6918)
  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_ITSN2-1/5
FEATURES             Location/Qualifiers
     source          1..6918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..29
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        52..129
                     /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             130..1212
                     /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(149..168)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1455..1460,1461..1476,1477..1483)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1491..1507
                     /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     1496..1518
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1592..1609
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1625..2278
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2236..2258
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2285..2308
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2321..2338
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2567..2614
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4207..4662)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4344..4365)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4556..4575
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4704..4726)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4812..4952
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4867..4886
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4967..4984)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5138..5725)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5896..6687,6688..6756))
                     /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     6519..6538
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6757..6861)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattcc cgacaccatc
       61 gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
      121 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
      181 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
      241 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
      301 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
      361 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
      421 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
      481 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
      541 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
      601 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
      661 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
      721 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
      781 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
      841 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
      901 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
      961 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     1021 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     1081 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     1141 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     1201 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg cacaattctc
     1261 atgtttgaca gcttatcatc gactgcacgg tgcaccaatg cttctggcgt caggcagcca
     1321 tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt gtcgctcaag
     1381 gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat
     1441 tctgaaatga gctgttgaca attaatcatc ggctcgtata atgtgtggaa ttgtgagcgg
     1501 ataacaattt cacacaggaa acagccagtc cgtttaggtg ttttcacgag cacttcacca
     1561 acaaggacca tagattatga aaatcgaaga acaccatcac catcaccatt ccagcggtaa
     1621 gcttatgtcc cctatactag gttattggaa aattaagggc cttgtgcaac ccactcgact
     1681 tcttttggaa tatcttgaag aaaaatatga agagcatttg tatgagcgcg atgaaggtga
     1741 taaatggcga aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat
     1801 tgatggtgat gttaaattaa cacagtctat ggccatcata cgttatatag ctgacaagca
     1861 caacatgttg ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt
     1921 tttggatatt agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa
     1981 agttgatttt cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca
     2041 taaaacatat ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct
     2101 tgatgttgtt ttatacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt
     2161 taaaaaacgt attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat
     2221 agcatggcct ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaatc
     2281 ggatctggaa gttctgttcc aggggcccct gtccagcggt ctggttccgc gtggttccgg
     2341 taccgcggcc cagccggcca tggcccaggg cgcgctggtt aactatcgtg ccctgtatcc
     2401 atttgaagcc cgtaaccatg atgaaatgtc ctttaactcc ggtgacatta tccaagtcga
     2461 tgaaaaaacc gttggtgaac cgggttggct gtatggttcc tttcagggta actttggttg
     2521 gtttccgtgc aactacgtcg aaaaaatgcc ggcggccgca taataaaccg atacaattaa
     2581 aggctccttt tggagccttt tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa
     2641 ttcctttagt tgttcctttc tattctcact ccgctgaaac tgttgaaagt tgtttagcaa
     2701 aaccccatac agaaaattca tttactaacg tctggaaaga cgacaaaact ttagatcgtt
     2761 acgctaacta tgagggctgt ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg
     2821 aaactcagtg tctagctaga gtggcggtgg ctctggttcc ggtgattttg attatgaaaa
     2881 gatggcaaac gctaataagg gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc
     2941 tgacgctaaa ggcaaacttg attctgtcgc tactgattac ggtgctgcta tcgatggttt
     3001 cattggtgac gtttccggcc ttgctaatgg taatggtgct actggtgatt ttgctggctc
     3061 taattcccaa atggctcaag tcggtgacgg tgataattca cctttaatga ataatttccg
     3121 tcaatattta ccttccctcc ctcaatcggt tgaatgtcgc ccttttgtct ttggcgctgg
     3181 taaaccatat gaattttcta ttgattgtga caaaataaac ttattccgtg gtgtctttgc
     3241 gtttctttta tatgttgcca cctttatgta tgtattttct acgtttgcta acatactgcg
     3301 taataaggag tcttaatcat gccagttctt ttggctagcg ccgccctata ccttgtctgc
     3361 ctccccgcgt tgcgtcgcgg tgcatggagc cgggccacct cgacctgaat ggaagccggc
     3421 ggcacctcgc taacggattc accactccaa gaattggagc caatcaattc ttgcggagaa
     3481 ctgtgaatgc gcaaaccaac ccttggcaga acatatccat cgcgtccgcc atctccagca
     3541 gccgcacgcg gcgcatctcg ggcagcgttg ggtcctggcc acgggtgcgc atgatcgtgc
     3601 tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat
     3661 caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa
     3721 caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc
     3781 cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac
     3841 ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc
     3901 gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat
     3961 cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca
     4021 gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg
     4081 cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg
     4141 atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagga
     4201 tccgcgaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     4261 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     4321 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     4381 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     4441 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     4501 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     4561 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     4621 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcgcggatc ctgcctcgcg
     4681 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     4741 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     4801 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     4861 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     4921 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     4981 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5041 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5101 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5161 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5221 gataccaggg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5281 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5341 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5401 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5461 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5521 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     5581 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5641 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5701 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5761 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5821 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5881 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5941 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6001 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6061 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6121 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6181 taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt
     6241 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6301 attgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6361 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6421 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6481 gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag
     6541 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6601 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6661 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6721 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6781 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6841 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     6901 cattattatc atgacatt
//
