LOCUS       sequence-192384-        6912 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            NCF1-2/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6912)
  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 6912)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_NCF1-2/2
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..45
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        68..145
                     /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             146..1228
                     /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(165..184)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1471..1476,1477..1492,1493..1499)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1507..1523
                     /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     1512..1534
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1608..1625
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1641..2294
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2252..2274
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2301..2324
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2337..2354
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2577..2624
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4217..4672)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4354..4375)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4566..4585
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4714..4736)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4822..4962
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4877..4896
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4977..4994)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5148..5735)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5906..6697,6698..6766))
                     /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     6529..6548
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6767..6871)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag
       61 aattcccgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga
      121 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
      181 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
      241 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
      301 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
      361 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
      421 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
      481 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
      541 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
      601 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
      661 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
      721 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
      781 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
      841 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
      901 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
      961 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     1021 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     1081 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     1141 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     1201 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     1261 attaggcaca attctcatgt ttgacagctt atcatcgact gcacggtgca ccaatgcttc
     1321 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata
     1381 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa
     1441 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt
     1501 gtggaattgt gagcggataa caatttcaca caggaaacag ccagtccgtt taggtgtttt
     1561 cacgagcact tcaccaacaa ggaccataga ttatgaaaat cgaagaacac catcaccatc
     1621 accattccag cggtaagctt atgtccccta tactaggtta ttggaaaatt aagggccttg
     1681 tgcaacccac tcgacttctt ttggaatatc ttgaagaaaa atatgaagag catttgtatg
     1741 agcgcgatga aggtgataaa tggcgaaaca aaaagtttga attgggtttg gagtttccca
     1801 atcttcctta ttatattgat ggtgatgtta aattaacaca gtctatggcc atcatacgtt
     1861 atatagctga caagcacaac atgttgggtg gttgtccaaa agagcgtgca gagatttcaa
     1921 tgcttgaagg agcggttttg gatattagat acggtgtttc gagaattgca tatagtaaag
     1981 actttgaaac tctcaaagtt gattttctta gcaagctacc tgaaatgctg aaaatgttcg
     2041 aagatcgttt atgtcataaa acatatttaa atggtgatca tgtaacccat cctgacttca
     2101 tgttgtatga cgctcttgat gttgttttat acatggaccc aatgtgcctg gatgcgttcc
     2161 caaaattagt ttgttttaaa aaacgtattg aagctatccc acaaattgat aagtacttga
     2221 aatccagcaa gtatatagca tggcctttgc agggctggca agccacgttt ggtggtggcg
     2281 accatcctcc aaaatcggat ctggaagttc tgttccaggg gcccctgtcc agcggtctgg
     2341 ttccgcgtgg ttccggtacc gcggcccagc cggccatggc ccagggcgcg ctggaaccat
     2401 acgttgccat taaagcctat accgccgtcg aaggtgatga agtttctctg ctggaaggtg
     2461 aagccgttga agtcattcat aaactgctgg atggttggtg ggttatccgt aaagatgatg
     2521 tcaccggtta ctttccgtcc atgtacttgc agaaatccgg tgcggccgca taataaaccg
     2581 atacaattaa aggctccttt tggagccttt tttttggaga ttttcaacgt gaaaaaatta
     2641 ttattcgcaa ttcctttagt tgttcctttc tattctcact ccgctgaaac tgttgaaagt
     2701 tgtttagcaa aaccccatac agaaaattca tttactaacg tctggaaaga cgacaaaact
     2761 ttagatcgtt acgctaacta tgagggctgt ctgtggaatg ctacaggcgt tgtagtttgt
     2821 actggtgacg aaactcagtg tctagctaga gtggcggtgg ctctggttcc ggtgattttg
     2881 attatgaaaa gatggcaaac gctaataagg gggctatgac cgaaaatgcc gatgaaaacg
     2941 cgctacagtc tgacgctaaa ggcaaacttg attctgtcgc tactgattac ggtgctgcta
     3001 tcgatggttt cattggtgac gtttccggcc ttgctaatgg taatggtgct actggtgatt
     3061 ttgctggctc taattcccaa atggctcaag tcggtgacgg tgataattca cctttaatga
     3121 ataatttccg tcaatattta ccttccctcc ctcaatcggt tgaatgtcgc ccttttgtct
     3181 ttggcgctgg taaaccatat gaattttcta ttgattgtga caaaataaac ttattccgtg
     3241 gtgtctttgc gtttctttta tatgttgcca cctttatgta tgtattttct acgtttgcta
     3301 acatactgcg taataaggag tcttaatcat gccagttctt ttggctagcg ccgccctata
     3361 ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc cgggccacct cgacctgaat
     3421 ggaagccggc ggcacctcgc taacggattc accactccaa gaattggagc caatcaattc
     3481 ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga acatatccat cgcgtccgcc
     3541 atctccagca gccgcacgcg gcgcatctcg ggcagcgttg ggtcctggcc acgggtgcgc
     3601 atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag
     3661 cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg
     3721 acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg
     3781 aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc
     3841 tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc
     3901 tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca
     3961 tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc
     4021 cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc
     4081 cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag
     4141 ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt
     4201 taccgcagga tccgcgaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     4261 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     4321 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     4381 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     4441 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     4501 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     4561 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     4621 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcgcggatc
     4681 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     4741 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     4801 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     4861 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     4921 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg
     4981 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5041 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5101 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5161 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5221 ggactataaa gataccaggg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5281 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5341 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5401 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5461 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5521 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5581 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5641 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5701 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5761 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5821 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5881 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5941 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6001 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6061 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6121 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6181 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac
     6241 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6301 gatcccccat attgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6361 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6421 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6481 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc
     6541 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6601 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6661 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6721 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6781 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6841 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6901 tctaagaaac ca
//
