LOCUS       sequence-67096-e        6508 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FX cloning E.coli expression vector with araBAD promoter and 
            C-terminal 3C protease cleavage site, GFP and 10x His tags.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6508)
  AUTHORS   Geertsma ER, Dutzler R
  TITLE     A versatile and efficient high-throughput cloning tool for 
            structural biology.
  JOURNAL   Biochemistry. 2011 Apr 19;50(15):3272-8. Epub 2011 Mar 25.
  PUBMED    21410291
REFERENCE   2  (bases 1 to 6508)
  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: 
            "Biochemistry."; date: "2011-04-19"; volume: "50(15)"; pages: 
            "3272-8. Epub 2011 Mar 25"
COMMENT     Sequence Label: pBXC3GH
FEATURES             Location/Qualifiers
     source          1..6508
                     /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"
     CDS             complement(370..675)
                     /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(472..491)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1017..1676)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1591..1610
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1730..1736,1737..1754,1755..1760))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(1785..1909)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1928..1948)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1952..1972)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1999..2022
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2029..2739
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2056..2084)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2679..2699
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2746..2775
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2791..2820
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2836..2853
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2909..2926)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3079..3165
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3257..3284
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3303..3394
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3395..3463,3464..4255)
                     /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(3613..3632)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4426..5014
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4915..4934
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5168..5185
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5200..5340
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5266..5285)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5426..5448
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5604..6482)
                     /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 gagttgaaga gcgacctgca gactggctgt gtataaggga
      361 gcctgacatt tatattcccc agaacatcag gttaatggcg tttttgatgt cattttcgcg
      421 gtggctgaga tcagccactt cttccccgat aacggagacc ggcacactgg ccatatcggt
      481 ggtcatcatg cgccagcttt catccccgat atgcaccacc gggtaaagtt cacgggagac
      541 tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcgcc cgggcgtgtc
      601 aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt tataggtgta
      661 aaccttaaac tgcatttcac cagcccctgt tctcgtcagc aaaagagccg ttcatttcaa
      721 taaaccgggc gacctcagcc atcccttcct gattttccgc tttccagcgt tcggcacgca
      781 gacgacgggc ttcattctgc atggttgtgc ttaccagacc ggagatattg acatcatata
      841 tgccttgagc aactgatagc tgtcgctgtc aactgtcact gtaatacgct gcttcatagc
      901 atacctcttt ttgacatact tcgggtatac atatcagtat atattcttat accgcaaaaa
      961 tcagcgcgca aatacgcata ctgttatctg gcttttagta agccggatcc acgcgtttac
     1021 gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg
     1081 aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct
     1141 tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg
     1201 tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca
     1261 ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat
     1321 atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca
     1381 gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg
     1441 tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag aatgtgaata
     1501 aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc
     1561 agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct
     1621 ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta
     1681 gcttccttag ctcctgaaaa tctcgacgga tcctaactca aaatccacac attatacgag
     1741 ccggaagcat aaagtgtaaa gcctggggtg cctaatgcgg ccgccatagt gactggatat
     1801 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
     1861 tatttatatc attttacgtt tctcgttcag cttttttgta caaacttgtg atatcgaatt
     1921 cgctagctac ctttctcttc ttttttggat ctacctttct cttctttttt ggggatccac
     1981 tagttcagct cttctgcatt agaagttttg tttcaaggtc cacaattcag caaaggagaa
     2041 gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac
     2101 aaattttctg tcagtggaga gggtgaaggt gatgctacat acggaaaact cacccttaaa
     2161 tttatttgca ctactggaaa actacctgtt ccttggccaa cacttgtcac tactctgacc
     2221 tatggtgttc aatgcttttc ccgttatccg gatcacatga aacggcatga ctttttcaag
     2281 agtgccatgc ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga tgacgggaac
     2341 tacaagacgc gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg tatcgagtta
     2401 aagggtattg attttaaaga agatggaaac attctcggac acaaactaga gtacaactat
     2461 aactcacaca atgtatacat cacggcagac aaacaaaaga atggaatcaa agctaacttc
     2521 aaaattcgcc acaacattga agatggttcc gttcaactag cagaccatta tcaacaaaat
     2581 actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc gacacaatct
     2641 gccctttcga aagatcccaa cgaaaagcgt gaccacatgg tccttcttga gtttgtaact
     2701 gctgctggga ttacacatgg catggatgag ctctacaaaa ctagtcatca tcatcaccat
     2761 catcatcacc atcattaagt cgaccatcta gaacaaaaac tcatctcaga agaggatctg
     2821 aatagcgccg tcgaccatca tcatcatcat cattgagttt aaacggtctc cagcttggct
     2881 gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg
     2941 gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc
     3001 cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag
     3061 tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     3121 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat
     3181 ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc
     3241 aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact
     3301 cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     3361 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     3421 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     3481 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     3541 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     3601 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact
     3661 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     3721 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     3781 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     3841 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     3901 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     3961 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     4021 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     4081 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     4141 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     4201 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     4261 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     4321 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     4381 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4441 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4501 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4561 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4621 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4681 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4741 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4801 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4861 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4921 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4981 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5041 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5101 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     5161 cgagcgcagc gagtcagtga gcgaggaagc ggaagtgccc ctgatgcggt attttctcct
     5221 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
     5281 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     5341 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5401 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5461 tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcataa
     5521 tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc tatgctactc cgtcaagccg
     5581 tcaattgtct gattcgttac caattatgac aacttgacgg ctacatcatt cactttttct
     5641 tcacaaccgg cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag
     5701 aaatagagtt gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg
     5761 gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta
     5821 atccctaact gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt
     5881 gcgacgctgg cgatatcaaa attgctgtct gccaggtgat cgctgatgta ctgacaagcc
     5941 tcgcgtaccc gattatccat cggtggatgg agcgactcgt taatcgcttc catgcgccgc
     6001 agtaacaatt gctcaagcag atttatcgcc agcagctccg aatagcgccc ttccccttgc
     6061 ccggcgttaa tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg
     6121 cgaaagaacc ccgtattggc aaatattgac ggccagttaa gccattcatg ccagtaggcg
     6181 cgcggacgaa agtaaaccca ctggtgatac cattcgcgag cctccggatg acgaccgtag
     6241 tgatgaatct ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac aaattctcgt
     6301 ccctgatttt tcaccacccc ctgaccgcga atggtgagat tgagaatata acctttcatt
     6361 cccagcggtc ggtcgataaa aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc
     6421 gccaccagat gggcattaaa cgagtatccc ggcagcaggg gatcattttg cgcttcagcc
     6481 atacttttca tactcccgcc attcagag
//
