LOCUS       sequence-258124-        6994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E.coli expression vector for FX cloning system, N-terminal pelB 
            signal sequence followed by 10xHisTag, maltose binding protein and 
            3C cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6994)
  AUTHORS   Zimmermann I, Egloff P, Hutter CA, Arnold FM, Stohler P, Bocquet N, 
            Hug MN, Huber S, Siegrist M, Hetemann L, Gera J, Gmur S, Spies P, 
            Gygax D, Geertsma ER, Dawson RJ, Seeger MA
  TITLE     Synthetic single domain antibodies for the conformational trapping 
            of membrane proteins.
  JOURNAL   Elife. 2018 May 24;7. pii: 34317. doi: 10.7554/eLife.34317.
  PUBMED    29792401
REFERENCE   2  (bases 1 to 6994)
  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: "Elife."; 
            date: "2018-05-24"; volume: "7. pii: 34317. doi"; pages: " 
            10.7554/eLife.34317"
COMMENT     Sequence Label: pBXNPHM3
FEATURES             Location/Qualifiers
     source          1..6994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..285
                     /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     208..227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     sig_peptide     319..384
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             424..453
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             457..1557
                     /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="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1531..1554
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1567..1590
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1639..1944)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1741..1760)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(2286..2945)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2860..2879
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2999..3005,3006..3023,3024..3029))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(3054..3178)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(3197..3217)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3221..3241)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3277..3306
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3322..3339
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3395..3412)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3565..3651
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3743..3770
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3789..3880
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3881..3949,3950..4741)
                     /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(4099..4118)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4912..5500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5401..5420
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5654..5671
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5686..5826
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5752..5771)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5912..5934
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6090..6968)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggcact
       61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca
      121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg
      181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg
      241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc
      301 taacaggagg aattaaccat gaaatacctg ctgccgaccg ctgctgctgg tctgctgctc
      361 ctcgctgccc agccggcgat ggccatggat atcggaatta attcggatcc gaattcgagc
      421 tcacatcacc atcaccatca ccatcaccat caccccatga aaatcgaaga aggtaaactg
      481 gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc
      541 gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc
      601 ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt
      661 ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac
      721 aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg
      781 atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa
      841 acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg
      901 atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat
      961 gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc
     1021 gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac
     1081 accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac
     1141 ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg
     1201 ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac
     1261 gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat
     1321 gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac
     1381 gaggaagagt tggcgaaaga tccacgtatt gccgccacta tggaaaacgc ccagaaaggt
     1441 gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg
     1501 atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactccg
     1561 ggtagcctgg aagttctgtt ccaggggccc agttgaagag cgacctgcag actggctgtg
     1621 tataagggag cctgacattt atattcccca gaacatcagg ttaatggcgt ttttgatgtc
     1681 attttcgcgg tggctgagat cagccacttc ttccccgata acggagaccg gcacactggc
     1741 catatcggtg gtcatcatgc gccagctttc atccccgata tgcaccaccg ggtaaagttc
     1801 acgggagact ttatctgaca gcagacgtgc actggccagg gggatcacca tccgtcgccc
     1861 gggcgtgtca ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt
     1921 ataggtgtaa accttaaact gcatttcacc agcccctgtt ctcgtcagca aaagagccgt
     1981 tcatttcaat aaaccgggcg acctcagcca tcccttcctg attttccgct ttccagcgtt
     2041 cggcacgcag acgacgggct tcattctgca tggttgtgct taccagaccg gagatattga
     2101 catcatatat gccttgagca actgatagct gtcgctgtca actgtcactg taatacgctg
     2161 cttcatagca tacctctttt tgacatactt cgggtataca tatcagtata tattcttata
     2221 ccgcaaaaat cagcgcgcaa atacgcatac tgttatctgg cttttagtaa gccggatcca
     2281 cgcgtttacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg
     2341 ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc
     2401 ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata
     2461 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac
     2521 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct
     2581 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa
     2641 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc
     2701 agctcaccgt ctttcattgc catacggaat tccggatgag cattcatcag gcgggcaaga
     2761 atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc
     2821 gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca
     2881 aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc
     2941 tccattttag cttccttagc tcctgaaaat ctcgacggat cctaactcaa aatccacaca
     3001 ttatacgagc cggaagcata aagtgtaaag cctggggtgc ctcatgcggc cgccatagtg
     3061 actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta
     3121 atatattgat atttatatca ttttacgttt ctcgttcagc ttttttgtac aaacttgtga
     3181 tatcgaattc gctagctacc tttctcttct tttttggatc tacctttctc ttcttttttg
     3241 gggatccact agttcagctc ttctgcataa taactagaac aaaaactcat ctcagaagag
     3301 gatctgaata gcgccgtcga ccatcatcat catcatcatt gagtttaaac ggtctccagc
     3361 ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca
     3421 gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc
     3481 ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg
     3541 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     3601 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
     3661 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
     3721 actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta
     3781 caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3841 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     3901 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3961 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4021 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4081 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     4141 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     4201 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     4261 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     4321 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     4381 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     4441 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     4501 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4561 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4621 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4681 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4741 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4801 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4861 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4921 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4981 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5041 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5101 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5161 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5221 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5281 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5341 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5401 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5461 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5521 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5581 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5641 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gtgcccctga tgcggtattt
     5701 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     5761 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     5821 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5881 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5941 ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat
     6001 gcataatgtg cctgtcaaat ggacgaagca gggattctgc aaaccctatg ctactccgtc
     6061 aagccgtcaa ttgtctgatt cgttaccaat tatgacaact tgacggctac atcattcact
     6121 ttttcttcac aaccggcacg gaactcgctc gggctggccc cggtgcattt tttaaatacc
     6181 cgcgagaaat agagttgatc gtcaaaacca acattgcgac cgacggtggc gataggcatc
     6241 cgggtggtgc tcaaaagcag cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag
     6301 acgctaatcc ctaactgctg gcggaaaaga tgtgacagac gcgacggcga caagcaaaca
     6361 tgctgtgcga cgctggcgat atcaaaattg ctgtctgcca ggtgatcgct gatgtactga
     6421 caagcctcgc gtacccgatt atccatcggt ggatggagcg actcgttaat cgcttccatg
     6481 cgccgcagta acaattgctc aagcagattt atcgccagca gctccgaata gcgcccttcc
     6541 ccttgcccgg cgttaatgat ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca
     6601 tccgggcgaa agaaccccgt attggcaaat attgacggcc agttaagcca ttcatgccag
     6661 taggcgcgcg gacgaaagta aacccactgg tgataccatt cgcgagcctc cggatgacga
     6721 ccgtagtgat gaatctctcc tggcgggaac agcaaaatat cacccggtcg gcaaacaaat
     6781 tctcgtccct gatttttcac caccccctga ccgcgaatgg tgagattgag aatataacct
     6841 ttcattccca gcggtcggtc gataaaaaaa tcgagataac cgttggcctc aatcggcgtt
     6901 aaacccgcca ccagatgggc attaaacgag tatcccggca gcaggggatc attttgcgct
     6961 tcagccatac ttttcatact cccgccattc agag
//
