LOCUS       sequence-335926-        5292 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of hybrid proteins in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5292)
  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 5292)
  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: pE5770
FEATURES             Location/Qualifiers
     source          1..5292
                     /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"
     CDS             44..67
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             143..1240
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1097..1106
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1214..1237
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1283..1303
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1416..1435)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1427..2242
                     /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             1427..2242
                     /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(1500..1519)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     oriT            3037..3146
                     /note="incP origin of transfer"
     CDS             join(3523..3591,3592..4383)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGVVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4511..5099
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5000..5019
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5253..5270
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg agaattcaag gagctttcat atgcaccatc atcatcacca
       61 tcaccatgga tctctgaacg acatcttcga agctcagaaa atcgaatggc atgaatacga
      121 tatcccaacg accgaaaacc tgaaaactga agaaggtaaa ctggtaatct ggattaacgg
      181 cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat
      241 taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac
      301 tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc
      361 tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac
      421 ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt
      481 atcgctgatt tataacaaag atttgctgcc gaacccgcca aaaacctggg aagagatccc
      541 ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga
      601 accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa
      661 cggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac
      721 cttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc
      781 agaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc
      841 caacatcgac accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca
      901 accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa
      961 agagctggca aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt
     1021 taataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa
     1081 agatccacgt attgccgcca ccatggaaaa cgcccagaaa ggtgaaatca tgccgaacat
     1141 cccgcagatg tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg
     1201 tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cgaacaacaa
     1261 caacaataac aataacaaca acgaaaacct gtattttcag ggatcctaag ggggggaaag
     1321 ccacgttgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg
     1381 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     1441 acgggaaacg tcttgctcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     1501 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     1561 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     1621 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     1681 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac
     1741 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     1801 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     1861 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     1921 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataagct
     1981 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     2041 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     2101 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     2161 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     2221 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat
     2281 tacgctgact tgacgggacg gcggctttgt tgaataaatc gaacttttgc tgagttgaag
     2341 gatcagatca cgcatcttcc cgacaacgca gaccgttccg tggcaaagca aaagttcaaa
     2401 atcaccaact ggtccaccta caacaaagct ctcatcaacc gtggctccct ctctagattc
     2461 aggtagttca cgttatgaga atgtgaaatt taacgatgta gcagttagtg gtggcttcta
     2521 tggtccaacg gcagcagagc ttggcggaca attccaccat aaatcagaaa atggcagtgt
     2581 aggtgctgtc tttggtgcaa aacaacaagt aaaaaaataa aagcttctgc ctggcggcct
     2641 tttgcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatc
     2701 gacatccgcc ctcaccgcca ggaacgcaac cgcagcctca tcacgccggc gcttcttggc
     2761 cgcgcgggat tcaacccact cggccagctc gtcggtgtag ctctttggta tcgtctctcg
     2821 cctgtcccct cagttcagta atttcctgca tttgcctgtt tccagtcggt agatattcca
     2881 caaaacagca gggaagcagc gcttttccgc tgcataaccc tgcttcgggg tcattatagc
     2941 gattttttcg gtatatccat cctttttcgc acgatataca ggattttgcc aaagggttcg
     3001 tgtagacttt ccttggtgta tccaacggcg tcagccgggc aggataggtg aagtaggccc
     3061 acccgcgagc gggtgttcct tcttcactgt cccttattcg cacctggcgg tgctcaacgg
     3121 gaatcctgct ctgcgaggct ggccggctac cgccggcgta acagatgagg gcaagcggat
     3181 ggctgatgaa accaagccaa ccaggaaggg cagcccacct atcaaggtgt actgccttcc
     3241 agacgaacga agagcgattg aggaaaaggc ggcggcggcc ggcatgagcc tgtcggccta
     3301 cctgctggcc gtcggccagg gctacaaaat cacgggcgtc gtggactatg agcacgtccg
     3361 cgagctggcc cgcatcaatg gcgacctggg ccgcctgggc ggcctgctga aactctggct
     3421 caccgacgac ccgcgcacgg cgcggttcgg tgatgccacg atcctcgccc tgctggcgaa
     3481 gatcgactct agctagacct gcaggattga aaaaggaaga gtatgagtat tcaacatttc
     3541 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3601 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3661 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3721 atgagcactt ttaaagttct gctatgtggc gtggtattat cccgtattga cgccgggcaa
     3781 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3841 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3901 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     3961 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4021 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     4081 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4141 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4201 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     4261 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4321 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4381 taaccctgca ggatgcattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4441 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4501 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4561 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4621 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     4681 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4741 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4801 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4861 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4921 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4981 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5041 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5101 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5161 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5221 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     5281 agcgcggccg ct
//
