LOCUS       sequence-336796-        5001 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 5001)
  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 5001)
  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: pE5772
FEATURES             Location/Qualifiers
     source          1..5001
                     /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"
     CDS             1491..2024
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     oriT            2746..2855
                     /note="incP origin of transfer"
     CDS             join(3232..3300,3301..4092)
                     /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      4220..4808
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4709..4728
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4962..4979
                     /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 ggatcctaaa agtagcgtat
     1321 gcgctcacgc aactggtcca gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc
     1381 ggttttcatg gcttgttatg actgtttttt tgtacagtct atgcctcggg catccaagca
     1441 gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg atgttacgca
     1501 gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaggt ggctcaagta
     1561 tgggcatcat tcgcacatgt aggctcggcc ctgaccaagt caaatccatg cgggctgctc
     1621 ttgatctttt cggtcgtgag ttcggagacg tagccaccta ctcccaacat cagccggact
     1681 ccgattacct cgggaacttg ctccgtagta agacattcat cgcgcttgct gccttcgacc
     1741 aagaagcggt tgttggcgct ctcgcggctt acgttctgcc caagtttgag cagccgcgta
     1801 gtgagatcta tatctatgat ctcgcagtct ccggcgagca ccggaggcag ggcattgcca
     1861 ccgcgctcat caatctcctc aagcatgagg ccaacgcgct tggtgcttat gtgatctacg
     1921 tgcaagcaga ttacggtgac gatcccgcag tggctctcta tacaaagttg ggcatacggg
     1981 aagaagtgat gcactttgat atcgacccaa gtaccgccac ctaaatgcat cagagatttt
     2041 gagacacaac gtggctttgt tgaataaatc gaacttttgc tgagttgaag gatcagatca
     2101 cgcatcttcc cgacaacgca gaccgttccg tggcaaagca aaagttcaaa atcaccaact
     2161 tctagattca ggtagttcac gttatgagaa tgtgaaattt aacgatgtag cagttagtgg
     2221 tggcttctat ggtccaacgg cagcagagct tggcggacaa ttccaccata aatcagaaaa
     2281 tggcagtgta ggtgctgtct ttggtgcaaa acaacaagta aaaaaataaa agcttctgcc
     2341 tggcggcctt ttgcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2401 ccgcatatcg acatccgccc tcaccgccag gaacgcaacc gcagcctcat cacgccggcg
     2461 cttcttggcc gcgcgggatt caacccactc ggccagctcg tcggtgtagc tctttggtat
     2521 cgtctctcgc ctgtcccctc agttcagtaa tttcctgcat ttgcctgttt ccagtcggta
     2581 gatattccac aaaacagcag ggaagcagcg cttttccgct gcataaccct gcttcggggt
     2641 cattatagcg attttttcgg tatatccatc ctttttcgca cgatatacag gattttgcca
     2701 aagggttcgt gtagactttc cttggtgtat ccaacggcgt cagccgggca ggataggtga
     2761 agtaggccca cccgcgagcg ggtgttcctt cttcactgtc ccttattcgc acctggcggt
     2821 gctcaacggg aatcctgctc tgcgaggctg gccggctacc gccggcgtaa cagatgaggg
     2881 caagcggatg gctgatgaaa ccaagccaac caggaagggc agcccaccta tcaaggtgta
     2941 ctgccttcca gacgaacgaa gagcgattga ggaaaaggcg gcggcggccg gcatgagcct
     3001 gtcggcctac ctgctggccg tcggccaggg ctacaaaatc acgggcgtcg tggactatga
     3061 gcacgtccgc gagctggccc gcatcaatgg cgacctgggc cgcctgggcg gcctgctgaa
     3121 actctggctc accgacgacc cgcgcacggc gcggttcggt gatgccacga tcctcgccct
     3181 gctggcgaag atcgactcta gctagacctg caggattgaa aaaggaagag tatgagtatt
     3241 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3301 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3361 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3421 tttccaatga tgagcacttt taaagttctg ctatgtggcg tggtattatc ccgtattgac
     3481 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3541 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3601 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3661 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3721 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     3781 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3841 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3901 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3961 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4021 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4081 aagcattggt aaccctgcag gatgcattta aaaggatcta ggtgaagatc ctttttgata
     4141 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4201 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4261 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4321 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     4381 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4441 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4501 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4561 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4621 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4681 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4741 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4801 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4861 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4921 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4981 aagcggaaga gcgcggccgc t
//
