LOCUS       sequence-335927-        5176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  production of tagged outer membrane protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5176)
  AUTHORS   Chaudhuri S, Ewasechko NF, Samaniego-Barron L, Fegan JE, Schryvers 
            AB
  TITLE     Design and Production of Hybrid Antigens for Targeting Integral 
            Outer Membrane Proteins in Gram-Negative Bacteria.
  JOURNAL   Methods Mol Biol. 2022;2414:115-140. doi: 
            10.1007/978-1-0716-1900-1_8.
  PUBMED    34784035
REFERENCE   2  (bases 1 to 5176)
  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: "Methods Mol
            Biol."; date: "2022"; volume: "2414:115-140. doi"; pages: " 
            10.1007/978-1-0716-1900-1_8"
COMMENT     Sequence Label: pE5771
FEATURES             Location/Qualifiers
     source          1..5176
                     /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"
     RBS             51..73
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     sig_peptide     81..146
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             153..176
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             201..314
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             327..347
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(460..479)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             471..1286
                     /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             471..1286
                     /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     complement(544..563)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             1677..1694
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1747..1765)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1761..1808
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1845..2300
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1932..1951)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2142..2163
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2326..2430
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2431..2499,2500..3291)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2649..2668)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3462..4050
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3951..3970
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4204..4221
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4236..4375
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4301..4320)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4461..4483
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4477..4668)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagtaataat tttgtttaac
       61 tttaagaagg agatatacat atgaaatacc tgctgccgac cgctgctgct ggtctgctgc
      121 tcctcgctgc ccagccggcg atggccatgg cccatcatca tcatcaccat caccatgaaa
      181 acctgtattt tcaggaattc atggacgaaa aaaccaccgg ttggcgtggt ggtcacgttg
      241 ttgaaggtct ggctggtgaa ctggaacagc tgcgtgctcg tctggaacac cacccgcagg
      301 gtcagcgtga accgagcagc ggcagagaaa acctgtattt tcagggatcc taaggggggg
      361 aaagccacgt tgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat
      421 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
      481 ttcaacggga aacgtcttgc tcgaggccgc gattaaattc caacatggat gctgatttat
      541 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt
      601 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
      661 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
      721 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggga
      781 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
      841 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
      901 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
      961 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     1021 agcttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     1081 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     1141 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     1201 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     1261 atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga
     1321 gcattacgct gacttgacgg gacggcggct ttgttgaata aatcgaactt ttgctgagtt
     1381 gaaggatcag atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt
     1441 caaaatcacc aactggtcca cctacaacaa agctctcatc aaccgtggct ccctctctag
     1501 attcaggtag ttcacgttat gagaatgtga aatttaacga tgtagcagtt agtggtggct
     1561 tctatggtcc aacggcagca gagcttggcg gacaattcca ccataaatca gaaaatggca
     1621 gtgtaggtgc tgtctttggt gcaaaacaac aagtaaaaaa ataaaagctt ctcgagcacc
     1681 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1741 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1801 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1861 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1921 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1981 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2041 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2101 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2161 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2221 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     2281 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2341 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2401 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     2461 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     2521 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     2581 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     2641 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     2701 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     2761 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     2821 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     2881 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     2941 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     3001 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3061 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3121 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     3181 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     3241 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     3301 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     3361 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     3421 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3481 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3541 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3601 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3661 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3721 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3781 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3841 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3901 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3961 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4021 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4081 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4141 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4201 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     4261 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     4321 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     4381 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4441 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4501 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     4561 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     4621 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     4681 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     4741 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     4801 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     4861 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     4921 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     4981 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     5041 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     5101 cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat
     5161 ctcgatcccg cgaaat
//
