LOCUS       sequence-215477-        7461 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of YWHAH_14-3-3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7461)
  AUTHORS   Teyra J, Kelil A, Jain S, Helmy M, Jajodia R, Hooda Y, Gu J, D'Cruz 
            AA, Nicholson SE, Min J, Sudol M, Kim PM, Bader GD, Sidhu SS
  TITLE     Large-scale survey and database of high affinity ligands for peptide
            recognition modules.
  JOURNAL   Mol Syst Biol. 2020 Dec;16(12):e9310. doi: 10.15252/msb.20199310.
  PUBMED    33438817
REFERENCE   2  (bases 1 to 7461)
  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"; journalName: "Mol Syst 
            Biol."; date: "2020-12"; volume: "16(12):e9310. doi"; pages: " 
            10.15252/msb.20199310"
COMMENT     Sequence Label: pHH0103_YWHAH_14-3-3
FEATURES             Location/Qualifiers
     source          1..7461
                     /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      1656..1703
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3296..3751)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3433..3454)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3645..3664
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3793..3815)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3901..4041
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3956..3975
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4056..4073)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4227..4814)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4985..5776,5777..5845))
                     /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     5608..5627
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5846..5950)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6018..6036
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6059..6136
                     /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             6137..7219
                     /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(6156..6175)
                     /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 cggccggcga tcgtgaacag ctgctgcaac gtgcacgcct ggctgaacag gcggaacgct
      961 atgatgacat ggcaagcgct atgaaagccg ttaccgaact gaacgaaccg ctgagtaacg
     1021 aagatcgtaa tctgctgtcc gtggcttaca aaaatgtggt tggtgcacgt cgcagctctt
     1081 ggcgtgttat tagttccatc gaacagaaaa ccatggctga tggtaatgag aaaaaactgg
     1141 aaaaagtcaa agcataccgt gaaaaaatcg aaaaagaact ggaaacggtt tgcaacgatg
     1201 tcctgtcact gctggacaaa ttcctgatca aaaactgtaa cgatttccag tacgaatcga
     1261 aagtcttcta cctgaaaatg aaaggcgact attaccgtta tctggccgaa gtggcatcag
     1321 gtgaaaagaa aaacagcgtc gtggaagcct ctgaagcggc ctacaaagaa gcattcgaaa
     1381 tcagcaaaga acagatgcaa ccgacgcatc cgatccgtct gggtctggca ctgaacttct
     1441 ctgtgttcta ttacgaaatc cagaacgccc cggaacaagc atgcctgctg gctaaacagg
     1501 cgttcgatga cgcgattgcc gaactggata ccctgaacga agattcatat aaagactcga
     1561 cgctgatcat gcaactgctg cgcgacaatc tgaccctgtg gacgtccgat cagcaagacg
     1621 aagaagcggg cgaaggtaac gcggccgcat aataaaccga tacaattaaa ggctcctttt
     1681 ggagcctttt ttttggagat tttcaacgtg aaaaaattat tattcgcaat tcctttagtt
     1741 gttcctttct attctcactc cgctgaaact gttgaaagtt gtttagcaaa accccataca
     1801 gaaaattcat ttactaacgt ctggaaagac gacaaaactt tagatcgtta cgctaactat
     1861 gagggctgtc tgtggaatgc tacaggcgtt gtagtttgta ctggtgacga aactcagtgt
     1921 ctagctagag tggcggtggc tctggttccg gtgattttga ttatgaaaag atggcaaacg
     1981 ctaataaggg ggctatgacc gaaaatgccg atgaaaacgc gctacagtct gacgctaaag
     2041 gcaaacttga ttctgtcgct actgattacg gtgctgctat cgatggtttc attggtgacg
     2101 tttccggcct tgctaatggt aatggtgcta ctggtgattt tgctggctct aattcccaaa
     2161 tggctcaagt cggtgacggt gataattcac ctttaatgaa taatttccgt caatatttac
     2221 cttccctccc tcaatcggtt gaatgtcgcc cttttgtctt tggcgctggt aaaccatatg
     2281 aattttctat tgattgtgac aaaataaact tattccgtgg tgtctttgcg tttcttttat
     2341 atgttgccac ctttatgtat gtattttcta cgtttgctaa catactgcgt aataaggagt
     2401 cttaatcatg ccagttcttt tggctagcgc cgccctatac cttgtctgcc tccccgcgtt
     2461 gcgtcgcggt gcatggagcc gggccacctc gacctgaatg gaagccggcg gcacctcgct
     2521 aacggattca ccactccaag aattggagcc aatcaattct tgcggagaac tgtgaatgcg
     2581 caaaccaacc cttggcagaa catatccatc gcgtccgcca tctccagcag ccgcacgcgg
     2641 cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct cctgtcgttg
     2701 aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc
     2761 gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg
     2821 gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt
     2881 atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta
     2941 ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc
     3001 gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt
     3061 atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct
     3121 tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc
     3181 agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca
     3241 gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcaggat ccgcgaaatt
     3301 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3361 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3421 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3481 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3541 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3601 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3661 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3721 gccgcgctta atgcgccgct acagggcgcg tcgcggatcc tgcctcgcgc gtttcggtga
     3781 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     3841 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     3901 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     3961 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
     4021 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4081 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4141 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4201 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4261 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccagggt
     4321 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4381 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4441 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4501 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4561 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4621 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4681 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4741 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4801 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4861 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4921 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4981 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5041 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5101 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5161 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5221 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5281 gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5341 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5401 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5461 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5521 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5581 gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag
     5641 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5701 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5761 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5821 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5881 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5941 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     6001 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaattcccga
     6061 caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     6121 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     6181 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     6241 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     6301 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     6361 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     6421 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     6481 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     6541 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     6601 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     6661 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     6721 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     6781 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     6841 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     6901 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     6961 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     7021 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     7081 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
     7141 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     7201 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     7261 aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag
     7321 gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc
     7381 gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg
     7441 caaatattct gaaatgagct g
//
