LOCUS       sequence-192249-        6918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            NEBL-1/1.
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_NEBL-1/1
FEATURES             Location/Qualifiers
     source          1..6918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     28..46
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        69..146
                     /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             147..1229
                     /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(166..185)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1472..1477,1478..1493,1494..1500)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1508..1524
                     /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     1513..1535
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1609..1626
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1642..2295
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2253..2275
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2302..2325
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2338..2355
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2584..2631
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4224..4679)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4361..4382)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4573..4592
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4721..4743)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4829..4969
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4884..4903
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4984..5001)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5155..5742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5913..6704,6705..6773))
                     /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     6536..6555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6774..6878)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa
       61 gaattcccga caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg
      121 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
      181 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
      241 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
      301 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
      361 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
      421 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
      481 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
      541 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
      601 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
      661 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
      721 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
      781 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
      841 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
      901 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
      961 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     1021 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     1081 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     1141 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     1201 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact
     1261 cattaggcac aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt
     1321 ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat
     1381 aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata
     1441 acggttctgg caaatattct gaaatgagct gttgacaatt aatcatcggc tcgtataatg
     1501 tgtggaattg tgagcggata acaatttcac acaggaaaca gccagtccgt ttaggtgttt
     1561 tcacgagcac ttcaccaaca aggaccatag attatgaaaa tcgaagaaca ccatcaccat
     1621 caccattcca gcggtaagct tatgtcccct atactaggtt attggaaaat taagggcctt
     1681 gtgcaaccca ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat
     1741 gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc
     1801 aatcttcctt attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt
     1861 tatatagctg acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca
     1921 atgcttgaag gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa
     1981 gactttgaaa ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc
     2041 gaagatcgtt tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc
     2101 atgttgtatg acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc
     2161 ccaaaattag tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg
     2221 aaatccagca agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc
     2281 gaccatcctc caaaatcgga tctggaagtt ctgttccagg ggcccctgtc cagcggtctg
     2341 gttccgcgtg gttccggtac cgcggcccag ccggccatgg cccagggcgc gctgcgtacc
     2401 tatcgtgcca tgtatgatta ctccgcccag gatgaagatg aagtctcctt tcgtgatggt
     2461 gattacattg tcaacgtcca gccgattgat gatggttgga tgtatggtac ggttcaacgt
     2521 accggtcgta ctggtatgct gccagccaac tacattgaat ttgtcaacgc ggccgcataa
     2581 taaaccgata caattaaagg ctccttttgg agcctttttt ttggagattt tcaacgtgaa
     2641 aaaattatta ttcgcaattc ctttagttgt tcctttctat tctcactccg ctgaaactgt
     2701 tgaaagttgt ttagcaaaac cccatacaga aaattcattt actaacgtct ggaaagacga
     2761 caaaacttta gatcgttacg ctaactatga gggctgtctg tggaatgcta caggcgttgt
     2821 agtttgtact ggtgacgaaa ctcagtgtct agctagagtg gcggtggctc tggttccggt
     2881 gattttgatt atgaaaagat ggcaaacgct aataaggggg ctatgaccga aaatgccgat
     2941 gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt
     3001 gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact
     3061 ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct
     3121 ttaatgaata atttccgtca atatttacct tccctccctc aatcggttga atgtcgccct
     3181 tttgtctttg gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta
     3241 ttccgtggtg tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttctacg
     3301 tttgctaaca tactgcgtaa taaggagtct taatcatgcc agttcttttg gctagcgccg
     3361 ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga
     3421 cctgaatgga agccggcggc acctcgctaa cggattcacc actccaagaa ttggagccaa
     3481 tcaattcttg cggagaactg tgaatgcgca aaccaaccct tggcagaaca tatccatcgc
     3541 gtccgccatc tccagcagcc gcacgcggcg catctcgggc agcgttgggt cctggccacg
     3601 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     3661 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
     3721 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     3781 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     3841 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     3901 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     3961 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     4021 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     4081 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     4141 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     4201 tgagctttac cgcaggatcc gcgaaattgt aagcgttaat attttgttaa aattcgcgtt
     4261 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     4321 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     4381 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     4441 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     4501 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     4561 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     4621 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     4681 gcggatcctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     4741 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     4801 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     4861 gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     4921 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     4981 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5041 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5101 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5161 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5221 cccgacagga ctataaagat accagggttt ccccctggaa gctccctcgt gcgctctcct
     5281 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5341 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5401 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5461 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5521 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5581 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5641 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5701 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5761 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5821 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5881 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5941 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6001 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6061 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6121 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6181 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg
     6241 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6301 gttacatgat cccccatatt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6361 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6421 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6481 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat
     6541 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6601 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6661 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6721 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6781 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6841 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6901 cctgacgtct aagaaacc
//
