LOCUS       sequence-191136-        6936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            NCF1-1/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6936)
  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 6936)
  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_NCF1-1/2
FEATURES             Location/Qualifiers
     source          1..6936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     17..35
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        58..135
                     /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             136..1218
                     /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(155..174)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1461..1466,1467..1482,1483..1489)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1497..1513
                     /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     1502..1524
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1598..1615
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1631..2284
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2242..2264
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2291..2314
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2327..2344
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2591..2638
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4231..4686)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4368..4389)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4580..4599
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4728..4750)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4836..4976
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4891..4910
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4991..5008)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5162..5749)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5920..6711,6712..6780))
                     /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     6543..6562
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6781..6885)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag aattcccgac
       61 accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
      121 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
      181 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
      241 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
      301 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
      361 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
      421 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
      481 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
      541 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
      601 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
      661 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
      721 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
      781 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
      841 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
      901 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat
      961 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg
     1021 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     1081 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa
     1141 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     1201 ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcaca
     1261 attctcatgt ttgacagctt atcatcgact gcacggtgca ccaatgcttc tggcgtcagg
     1321 cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg
     1381 ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa cggttctggc
     1441 aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt gtggaattgt
     1501 gagcggataa caatttcaca caggaaacag ccagtccgtt taggtgtttt cacgagcact
     1561 tcaccaacaa ggaccataga ttatgaaaat cgaagaacac catcaccatc accattccag
     1621 cggtaagctt atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac
     1681 tcgacttctt ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga
     1741 aggtgataaa tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta
     1801 ttatattgat ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga
     1861 caagcacaac atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg
     1921 agcggttttg gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac
     1981 tctcaaagtt gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt
     2041 atgtcataaa acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga
     2101 cgctcttgat gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt
     2161 ttgttttaaa aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa
     2221 gtatatagca tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc
     2281 aaaatcggat ctggaagttc tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg
     2341 ttccggtacc gcggcccagc cggccggcaa atcaacggca accgacatca cgggtccgat
     2401 cattctgcaa acctatcgcg ctatcgcaaa ctacgaaaaa acgagtggct ccgaaatggc
     2461 gctgagtacc ggcgatgtgg ttgaagtcgt tgaaaaaagc gaatctggtt ggtggttttg
     2521 ccagatgaaa gcgaaacgtg gttggattcc ggccagcttc ctggaaccgc tggacgcggc
     2581 cgcataataa accgatacaa ttaaaggctc cttttggagc cttttttttg gagattttca
     2641 acgtgaaaaa attattattc gcaattcctt tagttgttcc tttctattct cactccgctg
     2701 aaactgttga aagttgttta gcaaaacccc atacagaaaa ttcatttact aacgtctgga
     2761 aagacgacaa aactttagat cgttacgcta actatgaggg ctgtctgtgg aatgctacag
     2821 gcgttgtagt ttgtactggt gacgaaactc agtgtctagc tagagtggcg gtggctctgg
     2881 ttccggtgat tttgattatg aaaagatggc aaacgctaat aagggggcta tgaccgaaaa
     2941 tgccgatgaa aacgcgctac agtctgacgc taaaggcaaa cttgattctg tcgctactga
     3001 ttacggtgct gctatcgatg gtttcattgg tgacgtttcc ggccttgcta atggtaatgg
     3061 tgctactggt gattttgctg gctctaattc ccaaatggct caagtcggtg acggtgataa
     3121 ttcaccttta atgaataatt tccgtcaata tttaccttcc ctccctcaat cggttgaatg
     3181 tcgccctttt gtctttggcg ctggtaaacc atatgaattt tctattgatt gtgacaaaat
     3241 aaacttattc cgtggtgtct ttgcgtttct tttatatgtt gccaccttta tgtatgtatt
     3301 ttctacgttt gctaacatac tgcgtaataa ggagtcttaa tcatgccagt tcttttggct
     3361 agcgccgccc tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc
     3421 acctcgacct gaatggaagc cggcggcacc tcgctaacgg attcaccact ccaagaattg
     3481 gagccaatca attcttgcgg agaactgtga atgcgcaaac caacccttgg cagaacatat
     3541 ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat ctcgggcagc gttgggtcct
     3601 ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt
     3661 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct
     3721 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
     3781 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
     3841 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
     3901 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
     3961 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
     4021 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
     4081 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
     4141 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
     4201 acgctgatga gctttaccgc aggatccgcg aaattgtaag cgttaatatt ttgttaaaat
     4261 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     4321 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     4381 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     4441 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     4501 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     4561 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     4621 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     4681 gcgcgtcgcg gatcctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     4741 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     4801 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     4861 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     4921 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     4981 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5041 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5101 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5161 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5221 ggcgaaaccc gacaggacta taaagatacc agggtttccc cctggaagct ccctcgtgcg
     5281 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5341 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5401 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5461 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5521 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5581 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     5641 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5701 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5761 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5821 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5881 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5941 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6001 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6061 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6121 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6181 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg
     6241 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6301 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6361 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6421 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6481 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg
     6541 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6601 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6661 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6721 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6781 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6841 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6901 agtgccacct gacgtctaag aaaccattat tatcat
//
