LOCUS       sequence-336798-        4885 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 4885)
  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 4885)
  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: pE5773
FEATURES             Location/Qualifiers
     source          1..4885
                     /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"
     CDS             535..1068
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     CDS             1386..1403
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1456..1474)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1470..1517
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1554..2009
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1641..1660)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1851..1872
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2035..2139
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2140..2208,2209..3000)
                     /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(2358..2377)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3171..3759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3660..3679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3913..3930
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3945..4084
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4010..4029)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4170..4192
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4186..4377)
                     /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 taaaagtagc
      361 gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt aacggcgcag
      421 tggcggtttt catggcttgt tatgactgtt tttttgtaca gtctatgcct cgggcatcca
      481 agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc aacgatgtta
      541 cgcagcagca acgatgttac gcagcagggc agtcgcccta aaacaaagtt aggtggctca
      601 agtatgggca tcattcgcac atgtaggctc ggccctgacc aagtcaaatc catgcgggct
      661 gctcttgatc ttttcggtcg tgagttcgga gacgtagcca cctactccca acatcagccg
      721 gactccgatt acctcgggaa cttgctccgt agtaagacat tcatcgcgct tgctgccttc
      781 gaccaagaag cggttgttgg cgctctcgcg gcttacgttc tgcccaagtt tgagcagccg
      841 cgtagtgaga tctatatcta tgatctcgca gtctccggcg agcaccggag gcagggcatt
      901 gccaccgcgc tcatcaatct cctcaagcat gaggccaacg cgcttggtgc ttatgtgatc
      961 tacgtgcaag cagattacgg tgacgatccc gcagtggctc tctatacaaa gttgggcata
     1021 cgggaagaag tgatgcactt tgatatcgac ccaagtaccg ccacctaaat gcatcagaga
     1081 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag
     1141 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc
     1201 aacttctaga ttcaggtagt tcacgttatg agaatgtgaa atttaacgat gtagcagtta
     1261 gtggtggctt ctatggtcca acggcagcag agcttggcgg acaattccac cataaatcag
     1321 aaaatggcag tgtaggtgct gtctttggtg caaaacaaca agtaaaaaaa taaaagcttc
     1381 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     1441 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     1501 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     1561 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1621 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1681 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1741 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     1801 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     1861 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     1921 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     1981 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     2041 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2101 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2161 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2221 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2281 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2341 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     2401 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2461 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2521 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     2581 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     2641 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     2701 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     2761 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     2821 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     2881 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     2941 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3001 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3061 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3121 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3181 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3241 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3301 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3361 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3421 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3481 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3541 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3601 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3661 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3721 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3781 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3841 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3901 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3961 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     4021 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     4081 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4141 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4201 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     4261 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     4321 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     4381 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     4441 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     4501 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     4561 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     4621 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     4681 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     4741 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     4801 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct
     4861 gcccgagatc tcgatcccgc gaaat
//
