LOCUS       sequence-215048-        7464 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of YWHAB_14-3-3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7464)
  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 7464)
  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_YWHAB_14-3-3
FEATURES             Location/Qualifiers
     source          1..7464
                     /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      1659..1706
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3299..3754)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3436..3457)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3648..3667
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3796..3818)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3904..4044
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3959..3978
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4059..4076)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4230..4817)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4988..5779,5780..5848))
                     /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     5611..5630
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5849..5953)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6021..6039
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6062..6139
                     /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             6140..7222
                     /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(6159..6178)
                     /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 cggccatgac gatggataaa tcagaactgg tgcagaaagc aaaactggca gaacaggcgg
      961 aacgctacga cgatatggca gcagcaatga aggcggtcac cgaacagggc catgaactga
     1021 gcaacgaaga acgtaatctg ctgtctgttg catataaaaa cgtggttggt gctcgtcgca
     1081 gctcttggcg tgtcattagt tccatcgaac agaaaacgga acgcaatgaa aagaaacagc
     1141 aaatgggcaa agaataccgc gaaaagattg aagcagaact gcaagatatc tgcaacgacg
     1201 tgctggaact gctggataaa tatctgattc cgaatgctac ccagccggaa tctaaggtgt
     1261 tttatctgaa aatgaagggc gattatttcc gttacctgag cgaagtcgcc tctggtgaca
     1321 acaaacagac cacggtgtca aattcgcagc aagcgtacca agaagccttc gaaatcagta
     1381 aaaaggaaat gcagccgacc cacccgatcc gtctgggtct ggcgctgaac tttagcgttt
     1441 tctattacga aatcctgaat tcaccggaaa aagcctgttc gctggcaaag acggctttcg
     1501 atgaagcgat tgccgaactg gacaccctga acgaagaaag ttacaaagat tccacgctga
     1561 tcatgcaact gctgcgtgac aacctgaccc tgtggacgag cgaaaatcag ggcgatgaag
     1621 gtgacgcggg cgaaggtgaa aatgcggccg cataataaac cgatacaatt aaaggctcct
     1681 tttggagcct tttttttgga gattttcaac gtgaaaaaat tattattcgc aattccttta
     1741 gttgttcctt tctattctca ctccgctgaa actgttgaaa gttgtttagc aaaaccccat
     1801 acagaaaatt catttactaa cgtctggaaa gacgacaaaa ctttagatcg ttacgctaac
     1861 tatgagggct gtctgtggaa tgctacaggc gttgtagttt gtactggtga cgaaactcag
     1921 tgtctagcta gagtggcggt ggctctggtt ccggtgattt tgattatgaa aagatggcaa
     1981 acgctaataa gggggctatg accgaaaatg ccgatgaaaa cgcgctacag tctgacgcta
     2041 aaggcaaact tgattctgtc gctactgatt acggtgctgc tatcgatggt ttcattggtg
     2101 acgtttccgg ccttgctaat ggtaatggtg ctactggtga ttttgctggc tctaattccc
     2161 aaatggctca agtcggtgac ggtgataatt cacctttaat gaataatttc cgtcaatatt
     2221 taccttccct ccctcaatcg gttgaatgtc gcccttttgt ctttggcgct ggtaaaccat
     2281 atgaattttc tattgattgt gacaaaataa acttattccg tggtgtcttt gcgtttcttt
     2341 tatatgttgc cacctttatg tatgtatttt ctacgtttgc taacatactg cgtaataagg
     2401 agtcttaatc atgccagttc ttttggctag cgccgcccta taccttgtct gcctccccgc
     2461 gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga atggaagccg gcggcacctc
     2521 gctaacggat tcaccactcc aagaattgga gccaatcaat tcttgcggag aactgtgaat
     2581 gcgcaaacca acccttggca gaacatatcc atcgcgtccg ccatctccag cagccgcacg
     2641 cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg
     2701 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
     2761 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
     2821 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
     2881 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
     2941 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
     3001 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
     3061 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
     3121 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
     3181 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
     3241 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag gatccgcgaa
     3301 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     3361 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     3421 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     3481 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     3541 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     3601 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     3661 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     3721 cccgccgcgc ttaatgcgcc gctacagggc gcgtcgcgga tcctgcctcg cgcgtttcgg
     3781 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     3841 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     3901 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg
     3961 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     4021 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4081 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4141 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4201 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4261 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4321 ggtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4381 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4441 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4501 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4561 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4621 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     4681 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4741 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4801 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4861 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4921 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4981 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5041 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5101 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5161 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5221 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5281 tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5341 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5401 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5461 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5521 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5581 cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa
     5641 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5701 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5761 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5821 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5881 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5941 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     6001 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattcc
     6061 cgacaccatc gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     6121 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     6181 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     6241 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     6301 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     6361 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     6421 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     6481 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     6541 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     6601 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     6661 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     6721 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     6781 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     6841 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     6901 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     6961 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     7021 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     7081 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     7141 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     7201 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     7261 cacaattctc atgtttgaca gcttatcatc gactgcacgg tgcaccaatg cttctggcgt
     7321 caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt
     7381 gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc
     7441 tggcaaatat tctgaaatga gctg
//
