LOCUS       sequence-311624-        6187 bp    DNA     circular SYN 15-APR-2025
DEFINITION  ""Enhances the yields of soluble target proteins in E. coli 
            expression system"", ""Enable quick and efficient protein 
            purification with high purity and stability"".
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6187)
  TITLE     Truncated flagellin as a fusion partner for production of stably 
            folded recombinant proteins in E. coli
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6187)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pET-FleBt
FEATURES             Location/Qualifiers
     source          1..6187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     76..95
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     179..198
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        179..197
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    198..222
                     /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             237..259
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             278..295
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             305..322
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1160..1183
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1230..1247
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1300..1318)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1314..1361
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1398..1853
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1485..1504)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1695..1716
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1945..2760)
                     /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(1945..2760)
                     /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     2668..2687
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2752..2771
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2882..3470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3371..3390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3624..3641
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3656..3795
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3721..3740)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3881..3903
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3897..4088)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4897..5979)
                     /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     5941..5960
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5980..6057)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
       61 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
      121 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
      181 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
      241 tttaacttta agaaggagat ataccatggg cagcagccat catcatcatc atcacagcag
      301 cggcctggtg ccgcgcggca gccatatgac ttctaacatc aacggtttga ctgtggcagc
      361 acgtaatgcc aacgacggta tctcactgtc acagactgct gaaggcgcgt tgggcgaaat
      421 caacaacaac ttgcaacgtg tgcgtgacct gactgttcag gcgcaaaaca gctctaactc
      481 agcatctgat atcgactcca tccagtctga agttaaccag cgcatggaag aaatcaaccg
      541 cgtgaccaag caaactgatt tcaacggcat caaagtattg gataaccgta ccaagacaga
      601 ctcaagctac gatttccagg tcggttcgaa agataatgaa caaatcagca ttgcgattgg
      661 tgcaagttct ggctggaatc tggcgacagc caatgctgat ggtacttcat cagatactgt
      721 aaatacttat gctttcacca agaaagctgc acttgatact gcgcaaactg actatgatac
      781 tgcgaatact gcgtatttgg ctgcggttaa aagcggtgtt gctggtgata ttacgactac
      841 caaagccaca ctggatggta aaaacaccgc attagctacc gcagttaaag atgcaactgc
      901 cgttaatgaa gcggtaaatg gcaaggtgcg tacagttgcc gccaaaggtt ttgacgtgtt
      961 gaatggcacc gtcgctgctg atggtaaagc aaccggtacc acgccgttgg ctgatatcga
     1021 taaagcgctg aaagcggttg atacacagcg cagcgtattg ggtgcgtctc agaaccgttt
     1081 tgagtcaacc atcactaacc tgaacaatac cgtgaacaac ctgacttcag cccgtggcgg
     1141 cgctagcggt ggcggttctc tggaagttct gttccagggg cccggatccg aattcgagct
     1201 ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
     1261 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     1321 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1381 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1441 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1501 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     1561 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1621 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     1681 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     1741 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1801 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     1861 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1921 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     1981 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     2041 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     2101 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     2161 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     2221 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     2281 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     2341 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     2401 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     2461 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     2521 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     2581 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     2641 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     2701 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     2761 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     2821 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2881 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2941 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3001 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3061 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3121 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3181 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3241 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3301 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3361 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3421 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3481 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3541 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3601 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     3661 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     3721 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     3781 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3841 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3901 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     3961 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     4021 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     4081 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     4141 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     4201 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     4261 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     4321 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     4381 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     4441 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     4501 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     4561 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     4621 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     4681 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     4741 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     4801 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     4861 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4921 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4981 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     5041 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     5101 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     5161 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     5221 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     5281 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     5341 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     5401 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     5461 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     5521 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     5581 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     5641 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     5701 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     5761 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     5821 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     5881 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     5941 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     6001 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     6061 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     6121 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     6181 ccccggc
//
