LOCUS       sequence-193872-        6012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses easily crystallizable engineered 14-3-3 sigma variant 
            fused to the HSPB6 phosphorylatable peptide.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6012)
  AUTHORS   Sluchanko NN, Tugaeva KV, Greive SJ, Antson AA
  TITLE     Chimeric 14-3-3 proteins for unraveling interactions with 
            intrinsically disordered partners.
  JOURNAL   Sci Rep. 2017 Sep 20;7(1):12014. doi: 10.1038/s41598-017-12214-9.
  PUBMED    28931924
REFERENCE   2  (bases 1 to 6012)
  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: "Sci Rep."; 
            date: "2017-09-20"; volume: "7(1):12014. doi"; pages: " 
            10.1038/s41598-017-12214-9"
COMMENT     Sequence Label: pET28-his-3C_Clu3B6p
FEATURES             Location/Qualifiers
     source          1..6012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             94..111
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             112..135
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             877..894
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(947..965)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      961..1008
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1045..1500
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1132..1151)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1342..1363
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1592..2407)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1592..2407)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2315..2334
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2399..2418
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2529..3117
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3018..3037
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3271..3288
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3303..3442
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3368..3387)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3528..3550
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3544..3735)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4544..5626)
                     /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     5588..5607
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5627..5704)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5910..5929
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gaacatcatc atcatcatca tctggaggtg
      121 ctgttccaag gtcctcatat ggagagagcc agtctgatcc agaaggccaa gctggcagag
      181 caggccgaac gctatgagga catggcagcc ttcatgaaag gcgccgtgga gaagggcgag
      241 gagctctcct gcgaagagcg aaacctgctc tcagtagcct ataagaacgt ggtgggcggc
      301 cagagggctg cctggagggt gctgtccagt attgagcaga aaagcaacga ggagggatcg
      361 gcagcagcag ggcccgaggt gcgtgagtac cgggagaagg tggagactga gctccagggc
      421 gtgtgcgaca ccgtgctggg cctgctggac agccacctca tcaaggaggc cggggacgcc
      481 gagagccggg tcttctacct gaagatgaag ggtgactact accgctacct ggccgaggtg
      541 gccaccggtg acgacaagaa gcgcatcatt gactcagccc ggtcagccta ccaggaggcc
      601 atggacatca gcaagaagga gatgccgccc accaacccca tccgcctggg cctggccctg
      661 aacttttccg tcttccacta cgagatcgcc aacagccccg aggaggccat ctctctggcc
      721 aagaccactt tcgacgaggc catggctgat ctgcacaccc tcagcgagga ctcctacaaa
      781 gacagcaccc tcatcatgca gctgctgcga gacaacctga cactgtggac gggatcggga
      841 tccctgcgcc gcgctagcgc cccgttgtga ctcgagcacc accaccacca ccactgagat
      901 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
      961 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     1021 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1081 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1141 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1201 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1261 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1321 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1381 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1441 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     1501 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1561 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     1621 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     1681 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     1741 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     1801 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     1861 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     1921 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     1981 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     2041 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     2101 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     2161 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     2221 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     2281 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     2341 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     2401 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     2461 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2521 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2581 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2641 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     2701 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2761 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2821 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2881 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2941 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3001 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3061 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3121 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3181 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3241 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3301 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     3361 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     3421 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     3481 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3541 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     3601 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     3661 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     3721 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     3781 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     3841 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     3901 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     3961 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4021 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4081 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4141 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     4201 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     4261 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     4321 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     4381 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     4441 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     4501 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     4561 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4621 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     4681 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     4741 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     4801 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     4861 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     4921 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     4981 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     5041 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     5101 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     5161 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     5221 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     5281 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     5341 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     5401 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     5461 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     5521 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     5581 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     5641 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     5701 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     5761 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     5821 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     5881 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     5941 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     6001 atcccgcgaa at
//
