LOCUS       sequence-215049-        7467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of YWHAG_14-3-3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7467)
  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 7467)
  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_YWHAG_14-3-3
FEATURES             Location/Qualifiers
     source          1..7467
                     /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"
     CDS             complement(1500..1511)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     terminator      1662..1709
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3302..3757)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3439..3460)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3651..3670
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3799..3821)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3907..4047
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3962..3981
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4062..4079)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4233..4820)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4991..5782,5783..5851))
                     /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     5614..5633
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5852..5956)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6024..6042
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6065..6142
                     /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             6143..7225
                     /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(6162..6181)
                     /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 cggccatggt ggaccgtgaa caactggtgc aaaaagcccg tctggcggaa caagccgaac
      961 gctatgatga catggcagca gcaatgaaga atgttaccga actgaacgaa ccgctgagca
     1021 acgaagaacg taatctgctg tctgtggcat ataagaatgt ggttggcgct cgtcgcagct
     1081 cttggcgcgt tattagttcc atcgaacaga aaacgagtgc cgacggtaac gaaaagaaaa
     1141 ttgaaatggt ccgtgcgtat cgcgaaaaaa tcgaaaagga actggaagcc gtttgccagg
     1201 acgtcctgag cctgctggat aactacctga tcaagaactg tagtgaaacc caatacgaat
     1261 ccaaggtctt ttacctgaaa atgaagggcg attattaccg ttatctggca gaagtggcta
     1321 ccggtgaaaa acgcgcaacg gtcgtggaat catcggaaaa ggcatactca gaagctcatg
     1381 aaatttcgaa agaacatatg cagccgaccc acccgatccg tctgggtctg gcgctgaact
     1441 actctgtgtt ttattacgaa atccagaacg cgccggaaca agcctgccac ctggcgaaaa
     1501 cggccttcga tgacgcgatt gccgaactgg ataccctgaa cgaagacagc tacaaagatt
     1561 ctacgctgat catgcaactg ctgcgcgaca atctgaccct gtggacgagc gatcagcaag
     1621 atgacgatgg cggtgagggt aacaatgcgg ccgcataata aaccgataca attaaaggct
     1681 ccttttggag cctttttttt ggagattttc aacgtgaaaa aattattatt cgcaattcct
     1741 ttagttgttc ctttctattc tcactccgct gaaactgttg aaagttgttt agcaaaaccc
     1801 catacagaaa attcatttac taacgtctgg aaagacgaca aaactttaga tcgttacgct
     1861 aactatgagg gctgtctgtg gaatgctaca ggcgttgtag tttgtactgg tgacgaaact
     1921 cagtgtctag ctagagtggc ggtggctctg gttccggtga ttttgattat gaaaagatgg
     1981 caaacgctaa taagggggct atgaccgaaa atgccgatga aaacgcgcta cagtctgacg
     2041 ctaaaggcaa acttgattct gtcgctactg attacggtgc tgctatcgat ggtttcattg
     2101 gtgacgtttc cggccttgct aatggtaatg gtgctactgg tgattttgct ggctctaatt
     2161 cccaaatggc tcaagtcggt gacggtgata attcaccttt aatgaataat ttccgtcaat
     2221 atttaccttc cctccctcaa tcggttgaat gtcgcccttt tgtctttggc gctggtaaac
     2281 catatgaatt ttctattgat tgtgacaaaa taaacttatt ccgtggtgtc tttgcgtttc
     2341 ttttatatgt tgccaccttt atgtatgtat tttctacgtt tgctaacata ctgcgtaata
     2401 aggagtctta atcatgccag ttcttttggc tagcgccgcc ctataccttg tctgcctccc
     2461 cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc tgaatggaag ccggcggcac
     2521 ctcgctaacg gattcaccac tccaagaatt ggagccaatc aattcttgcg gagaactgtg
     2581 aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt ccgccatctc cagcagccgc
     2641 acgcggcgca tctcgggcag cgttgggtcc tggccacggg tgcgcatgat cgtgctcctg
     2701 tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg
     2761 atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca
     2821 tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc
     2881 accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca
     2941 tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc
     3001 cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta
     3061 acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat
     3121 cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc
     3181 tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa
     3241 caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg caggatccgc
     3301 gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     3361 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     3421 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     3481 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     3541 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     3601 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     3661 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     3721 acacccgccg cgcttaatgc gccgctacag ggcgcgtcgc ggatcctgcc tcgcgcgttt
     3781 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     3841 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     3901 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     3961 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga
     4021 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     4081 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4141 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4201 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4261 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4321 cagggtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4381 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4441 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4501 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4561 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4621 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     4681 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4741 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4801 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4861 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4921 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4981 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5041 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5101 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5161 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5221 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5281 gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta
     5341 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5401 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5461 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5521 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5581 gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt
     5641 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5701 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5761 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5821 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5881 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5941 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     6001 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat
     6061 tcccgacacc atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     6121 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     6181 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     6241 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     6301 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     6361 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     6421 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     6481 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     6541 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     6601 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     6661 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     6721 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     6781 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     6841 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     6901 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata
     6961 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     7021 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     7081 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     7141 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     7201 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     7261 aggcacaatt ctcatgtttg acagcttatc atcgactgca cggtgcacca atgcttctgg
     7321 cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt
     7381 cgtgtcgctc aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg
     7441 ttctggcaaa tattctgaaa tgagctg
//
