LOCUS       sequence-201858-        7104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            NCF2-2/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7104)
  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 7104)
  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_NCF2-2/2
FEATURES             Location/Qualifiers
     source          1..7104
                     /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      1299..1346
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2939..3394)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3076..3097)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3288..3307
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3436..3458)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3544..3684
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3599..3618
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3699..3716)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3870..4457)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4628..5419,5420..5488))
                     /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     5251..5270
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5489..5593)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5661..5679
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5702..5779
                     /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             5780..6862
                     /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(5799..5818)
                     /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 ctgtctcaag ttgaagccct gttctcttat gaagccaccc
      961 agccggaaga tttggaattt caggaaggtg acattatcct ggtcctgtcc aaagttaacg
     1021 aagaatggct ggaaggcgaa tgtaaaggta aagtcggtat cttcccgaaa gttttcgttg
     1081 aagattgcgc cgcggccgcg gaacgtgggg taacatagtt acgtggaaac ataccggtct
     1141 gaccatgaca tgcacctttc caccaattcg gatcggagtt atccatgaca tgaatgaaat
     1201 caccacgacg aaaacccagt tcaccatctt cctgcggatc aaaatcaaac agggcctgaa
     1261 cataggtcag cgcgccctgg gccatggccg cataataaac cgatacaatt aaaggctcct
     1321 tttggagcct tttttttgga gattttcaac gtgaaaaaat tattattcgc aattccttta
     1381 gttgttcctt tctattctca ctccgctgaa actgttgaaa gttgtttagc aaaaccccat
     1441 acagaaaatt catttactaa cgtctggaaa gacgacaaaa ctttagatcg ttacgctaac
     1501 tatgagggct gtctgtggaa tgctacaggc gttgtagttt gtactggtga cgaaactcag
     1561 tgtctagcta gagtggcggt ggctctggtt ccggtgattt tgattatgaa aagatggcaa
     1621 acgctaataa gggggctatg accgaaaatg ccgatgaaaa cgcgctacag tctgacgcta
     1681 aaggcaaact tgattctgtc gctactgatt acggtgctgc tatcgatggt ttcattggtg
     1741 acgtttccgg ccttgctaat ggtaatggtg ctactggtga ttttgctggc tctaattccc
     1801 aaatggctca agtcggtgac ggtgataatt cacctttaat gaataatttc cgtcaatatt
     1861 taccttccct ccctcaatcg gttgaatgtc gcccttttgt ctttggcgct ggtaaaccat
     1921 atgaattttc tattgattgt gacaaaataa acttattccg tggtgtcttt gcgtttcttt
     1981 tatatgttgc cacctttatg tatgtatttt ctacgtttgc taacatactg cgtaataagg
     2041 agtcttaatc atgccagttc ttttggctag cgccgcccta taccttgtct gcctccccgc
     2101 gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga atggaagccg gcggcacctc
     2161 gctaacggat tcaccactcc aagaattgga gccaatcaat tcttgcggag aactgtgaat
     2221 gcgcaaacca acccttggca gaacatatcc atcgcgtccg ccatctccag cagccgcacg
     2281 cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg
     2341 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
     2401 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
     2461 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
     2521 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
     2581 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
     2641 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
     2701 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
     2761 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
     2821 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
     2881 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag gatccgcgaa
     2941 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     3001 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     3061 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     3121 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     3181 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     3241 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     3301 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     3361 cccgccgcgc ttaatgcgcc gctacagggc gcgtcgcgga tcctgcctcg cgcgtttcgg
     3421 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     3481 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     3541 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg
     3601 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     3661 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3721 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3781 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3841 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3901 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3961 ggtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4021 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4081 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4141 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4201 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4261 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     4321 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4381 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4441 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4501 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4561 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4621 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4681 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4741 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4801 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4861 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4921 tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4981 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atattgtgca
     5041 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5101 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5161 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5221 cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa
     5281 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5341 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5401 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5461 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5521 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5581 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     5641 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattcc
     5701 cgacaccatc gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     5761 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     5821 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     5881 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     5941 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     6001 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     6061 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     6121 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     6181 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     6241 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     6301 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     6361 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     6421 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     6481 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     6541 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     6601 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     6661 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     6721 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     6781 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     6841 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     6901 cacaattctc atgtttgaca gcttatcatc gactgcacgg tgcaccaatg cttctggcgt
     6961 caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt
     7021 gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc
     7081 tggcaaatat tctgaaatga gctg
//
