LOCUS       sequence-258939-        5260 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Avi-tagged MBP in E.coli, to use as positive control in 
            ELISA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5260)
  AUTHORS   Zimmermann I, Egloff P, Hutter CAJ, Kuhn BT, Brauer P, Newstead S, 
            Dawson RJP, Geertsma ER, Seeger MA
  TITLE     Generation of synthetic nanobodies against delicate proteins.
  JOURNAL   Nat Protoc. 2020 Apr 8. pii: 10.1038/s41596-020-0304-x. doi: 
            10.1038/s41596-020-0304-x.
  PUBMED    32269381
REFERENCE   2  (bases 1 to 5260)
  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: "Nat Protoc.
            2020 Apr 8. pii: 10.1038/s41596-020-0304-x. doi: 
            10.1038/s41596-020-0304-x."
COMMENT     Sequence Label: pBXNH3CA_MBP
FEATURES             Location/Qualifiers
     source          1..5260
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             68..97
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             98..121
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             125..1222
                     /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="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1196..1219
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1235..1279
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1289..1318
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1334..1351
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1407..1424)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1577..1663
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1755..1782
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1801..1892
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1893..1961,1962..2753)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2111..2130)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2924..3512
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3413..3432
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3666..3683
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3698..3838
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3764..3783)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3924..3946
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4102..4980)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        join(5007..5260,1..31)
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     5214..5233
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
ORIGIN
        1 tttttatcgc aactctctac tgtttctcca tacccgtttt ttgggctaac aggaggaatt
       61 aaccatgcac catcatcatc accatcatca tcatcattta gaagttttgt ttcaaggtcc
      121 aagtaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg
      181 tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca
      241 tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat
      301 tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat
      361 caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta
      421 caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa
      481 agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact
      541 gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc
      601 gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa
      661 agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat
      721 taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa
      781 aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa
      841 agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt
      901 tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt
      961 cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct
     1021 gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc
     1081 cactatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt
     1141 ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga
     1201 agccctgaaa gacgcgcaga ctccgggtag cgcaggtctg aacgatatct tcgaagctca
     1261 gaaaatcgaa tggcacgaat aataactaga acaaaaactc atctcagaag aggatctgaa
     1321 tagcgccgtc gaccatcatc atcatcatca ttgagtttaa acggtctcca gcttggctgt
     1381 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt
     1441 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     1501 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta
     1561 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1621 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt
     1681 gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag
     1741 gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct
     1801 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1861 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1921 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1981 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2041 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2101 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2161 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2221 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2281 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2341 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2401 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     2461 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2521 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2581 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2641 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2701 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2761 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2821 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2881 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2941 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3001 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3061 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3121 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3181 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3241 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3301 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3361 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3421 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3481 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3541 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3601 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3661 agcgcagcga gtcagtgagc gaggaagcgg aagtgcccct gatgcggtat tttctcctta
     3721 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
     3781 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     3841 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3901 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3961 accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc atgcataatg
     4021 tgcctgtcaa atggacgaag cagggattct gcaaacccta tgctactccg tcaagccgtc
     4081 aattgtctga ttcgttacca attatgacaa cttgacggct acatcattca ctttttcttc
     4141 acaaccggca cggaactcgc tcgggctggc cccggtgcat tttttaaata cccgcgagaa
     4201 atagagttga tcgtcaaaac caacattgcg accgacggtg gcgataggca tccgggtggt
     4261 gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg cgccagctta agacgctaat
     4321 ccctaactgc tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa catgctgtgc
     4381 gacgctggcg atatcaaaat tgctgtctgc caggtgatcg ctgatgtact gacaagcctc
     4441 gcgtacccga ttatccatcg gtggatggag cgactcgtta atcgcttcca tgcgccgcag
     4501 taacaattgc tcaagcagat ttatcgccag cagctccgaa tagcgccctt ccccttgccc
     4561 ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt catccgggcg
     4621 aaagaacccc gtattggcaa atattgacgg ccagttaagc cattcatgcc agtaggcgcg
     4681 cggacgaaag taaacccact ggtgatacca ttcgcgagcc tccggatgac gaccgtagtg
     4741 atgaatctct cctggcggga acagcaaaat atcacccggt cggcaaacaa attctcgtcc
     4801 ctgatttttc accaccccct gaccgcgaat ggtgagattg agaatataac ctttcattcc
     4861 cagcggtcgg tcgataaaaa aatcgagata accgttggcc tcaatcggcg ttaaacccgc
     4921 caccagatgg gcattaaacg agtatcccgg cagcagggga tcattttgcg cttcagccat
     4981 acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg
     5041 ccgtcactgc gtcttttact ggcacttctc gctaaccaaa ccggtaaccc cgcttattaa
     5101 aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta
     5161 taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat
     5221 agcattttta tccataagat tagcggatcc tacctgacgc
//
