LOCUS       sequence-202036-        7141 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            NCF2-1/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7141)
  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 7141)
  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_NCF2-1/2
FEATURES             Location/Qualifiers
     source          1..7141
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             867..884
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      1336..1383
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2976..3431)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3113..3134)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3325..3344
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3473..3495)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3581..3721
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3636..3655
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3736..3753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3907..4494)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4665..5456,5457..5525))
                     /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     5288..5307
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5526..5630)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5698..5716
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5739..5816
                     /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             5817..6899
                     /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(5836..5855)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc
      901 cggccatggc ccagggcgcg ctggaagcac atcgtgtcct gtttggtttt gttccggaaa
      961 ccaaagaaga actgcaagtc atgccgggta acattgtttt cgttctgaaa aaaggtaacg
     1021 ataactgggc caccgttatg tttaacggtc agaaaggtct ggttccatgc aactatctgg
     1081 aaccggttga agcggccgcg gaattggtaa cggactatgt tcgtgaatta ccagcggaat
     1141 atgttcgtga attaccagcg gccgcacgga caatttcaac gtaggaaacc atacaacgcg
     1201 ggaaaccata caacgcggcc gcggaattgg taacggatgt tcgtgaatta ccagcggatg
     1261 ttcgtgaatt accagcggcc gcacggacaa tttcaacgtg gctttctgaa accatacaac
     1321 gcggccgcat aataaaccga tacaattaaa ggctcctttt ggagcctttt ttttggagat
     1381 tttcaacgtg aaaaaattat tattcgcaat tcctttagtt gttcctttct attctcactc
     1441 cgctgaaact gttgaaagtt gtttagcaaa accccataca gaaaattcat ttactaacgt
     1501 ctggaaagac gacaaaactt tagatcgtta cgctaactat gagggctgtc tgtggaatgc
     1561 tacaggcgtt gtagtttgta ctggtgacga aactcagtgt ctagctagag tggcggtggc
     1621 tctggttccg gtgattttga ttatgaaaag atggcaaacg ctaataaggg ggctatgacc
     1681 gaaaatgccg atgaaaacgc gctacagtct gacgctaaag gcaaacttga ttctgtcgct
     1741 actgattacg gtgctgctat cgatggtttc attggtgacg tttccggcct tgctaatggt
     1801 aatggtgcta ctggtgattt tgctggctct aattcccaaa tggctcaagt cggtgacggt
     1861 gataattcac ctttaatgaa taatttccgt caatatttac cttccctccc tcaatcggtt
     1921 gaatgtcgcc cttttgtctt tggcgctggt aaaccatatg aattttctat tgattgtgac
     1981 aaaataaact tattccgtgg tgtctttgcg tttcttttat atgttgccac ctttatgtat
     2041 gtattttcta cgtttgctaa catactgcgt aataaggagt cttaatcatg ccagttcttt
     2101 tggctagcgc cgccctatac cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc
     2161 gggccacctc gacctgaatg gaagccggcg gcacctcgct aacggattca ccactccaag
     2221 aattggagcc aatcaattct tgcggagaac tgtgaatgcg caaaccaacc cttggcagaa
     2281 catatccatc gcgtccgcca tctccagcag ccgcacgcgg cgcatctcgg gcagcgttgg
     2341 gtcctggcca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg
     2401 gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg
     2461 ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc
     2521 gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
     2581 aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg
     2641 accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca
     2701 acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg
     2761 tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac
     2821 caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct
     2881 tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca
     2941 cgaccacgct gatgagcttt accgcaggat ccgcgaaatt gtaagcgtta atattttgtt
     3001 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     3061 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     3121 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     3181 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3241 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3301 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3361 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3421 acagggcgcg tcgcggatcc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     3481 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     3541 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     3601 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     3661 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     3721 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3781 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3841 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3901 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3961 gaggtggcga aacccgacag gactataaag ataccagggt ttccccctgg aagctccctc
     4021 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4081 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4141 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4201 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4261 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4321 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     4381 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4441 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4501 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4561 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4621 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4681 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4741 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4801 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4861 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4921 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4981 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5041 cgatcaaggc gagttacatg atcccccata ttgtgcaaaa aagcggttag ctccttcggt
     5101 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5161 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5221 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5281 acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5341 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5401 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5461 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5521 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5581 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5641 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     5701 aggcgtatca cgaggccctt tcgtcttcaa gaattcccga caccatcgaa tggtgcaaaa
     5761 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     5821 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     5881 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     5941 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     6001 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     6061 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     6121 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     6181 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     6241 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     6301 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     6361 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     6421 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     6481 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     6541 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     6601 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     6661 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     6721 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     6781 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     6841 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     6901 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac aattctcatg tttgacagct
     6961 tatcatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
     7021 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
     7081 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
     7141 g
//
