LOCUS       sequence-67099-e        6556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FX cloning E.coli expression vector with araBAD promoter, N-terminal
            Avi-tag, 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 6556)
  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 6556)
  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: pBXNAC3GH
FEATURES             Location/Qualifiers
     source          1..6556
                     /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             325..369
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             complement(418..723)
                     /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(520..539)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1065..1724)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1639..1658
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1778..1784,1785..1802,1803..1808))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(1833..1957)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1976..1996)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2000..2020)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2047..2070
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2077..2787
                     /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(2104..2132)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2727..2747
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2794..2823
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2839..2868
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2884..2901
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2957..2974)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3127..3213
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3305..3332
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3351..3442
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3443..3511,3512..4303)
                     /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(3661..3680)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4474..5062
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4963..4982
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5216..5233
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5248..5388
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5314..5333)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5474..5496
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5652..6530)
                     /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 ggctggtctg aacgatatct tcgaagctca gaaaatcgaa
      361 tggcacgaaa gttgaagagc gacctgcaga ctggctgtgt ataagggagc ctgacattta
      421 tattccccag aacatcaggt taatggcgtt tttgatgtca ttttcgcggt ggctgagatc
      481 agccacttct tccccgataa cggagaccgg cacactggcc atatcggtgg tcatcatgcg
      541 ccagctttca tccccgatat gcaccaccgg gtaaagttca cgggagactt tatctgacag
      601 cagacgtgca ctggccaggg ggatcaccat ccgtcgcccg ggcgtgtcaa taatatcact
      661 ctgtacatcc acaaacagac gataacggct ctctctttta taggtgtaaa ccttaaactg
      721 catttcacca gcccctgttc tcgtcagcaa aagagccgtt catttcaata aaccgggcga
      781 cctcagccat cccttcctga ttttccgctt tccagcgttc ggcacgcaga cgacgggctt
      841 cattctgcat ggttgtgctt accagaccgg agatattgac atcatatatg ccttgagcaa
      901 ctgatagctg tcgctgtcaa ctgtcactgt aatacgctgc ttcatagcat acctcttttt
      961 gacatacttc gggtatacat atcagtatat attcttatac cgcaaaaatc agcgcgcaaa
     1021 tacgcatact gttatctggc ttttagtaag ccggatccac gcgtttacgc cccgccctgc
     1081 cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa
     1141 acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat
     1201 ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa
     1261 ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta
     1321 gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac
     1381 tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg
     1441 aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc
     1501 atacggaatt ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa
     1561 aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc
     1621 tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat
     1681 tgggatatat caacggtggt atatccagtg atttttttct ccattttagc ttccttagct
     1741 cctgaaaatc tcgacggatc ctaactcaaa atccacacat tatacgagcc ggaagcataa
     1801 agtgtaaagc ctggggtgcc taatgcggcc gccatagtga ctggatatgt tgtgttttac
     1861 agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat
     1921 tttacgtttc tcgttcagct tttttgtaca aacttgtgat atcgaattcg ctagctacct
     1981 ttctcttctt ttttggatct acctttctct tcttttttgg ggatccacta gttcagctct
     2041 tctgcattag aagttttgtt tcaaggtcca caattcagca aaggagaaga acttttcact
     2101 ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc
     2161 agtggagagg gtgaaggtga tgctacatac ggaaaactca cccttaaatt tatttgcact
     2221 actggaaaac tacctgttcc ttggccaaca cttgtcacta ctctgaccta tggtgttcaa
     2281 tgcttttccc gttatccgga tcacatgaaa cggcatgact ttttcaagag tgccatgccc
     2341 gaaggttatg tacaggaacg cactatatct ttcaaagatg acgggaacta caagacgcgt
     2401 gctgaagtca agtttgaagg tgataccctt gttaatcgta tcgagttaaa gggtattgat
     2461 tttaaagaag atggaaacat tctcggacac aaactagagt acaactataa ctcacacaat
     2521 gtatacatca cggcagacaa acaaaagaat ggaatcaaag ctaacttcaa aattcgccac
     2581 aacattgaag atggttccgt tcaactagca gaccattatc aacaaaatac tccaattggc
     2641 gatggccctg tccttttacc agacaaccat tacctgtcga cacaatctgc cctttcgaaa
     2701 gatcccaacg aaaagcgtga ccacatggtc cttcttgagt ttgtaactgc tgctgggatt
     2761 acacatggca tggatgagct ctacaaaact agtcatcatc atcaccatca tcatcaccat
     2821 cattaagtcg accatctaga acaaaaactc atctcagaag aggatctgaa tagcgccgtc
     2881 gaccatcatc atcatcatca ttgagtttaa acggtctcca gcttggctgt tttggcggat
     2941 gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac
     3001 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     3061 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     3121 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     3181 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
     3241 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt
     3301 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt
     3361 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3421 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3481 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3541 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3601 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3661 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     3721 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     3781 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3841 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3901 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3961 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     4021 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     4081 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     4141 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     4201 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     4261 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4321 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4381 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4441 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4501 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4561 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4621 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4681 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4741 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4801 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4861 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4921 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4981 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5041 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5101 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5161 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     5221 gtcagtgagc gaggaagcgg aagtgcccct gatgcggtat tttctcctta cgcatctgtg
     5281 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5341 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     5401 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5461 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5521 gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc atgcataatg tgcctgtcaa
     5581 atggacgaag cagggattct gcaaacccta tgctactccg tcaagccgtc aattgtctga
     5641 ttcgttacca attatgacaa cttgacggct acatcattca ctttttcttc acaaccggca
     5701 cggaactcgc tcgggctggc cccggtgcat tttttaaata cccgcgagaa atagagttga
     5761 tcgtcaaaac caacattgcg accgacggtg gcgataggca tccgggtggt gctcaaaagc
     5821 agcttcgcct ggctgatacg ttggtcctcg cgccagctta agacgctaat ccctaactgc
     5881 tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa catgctgtgc gacgctggcg
     5941 atatcaaaat tgctgtctgc caggtgatcg ctgatgtact gacaagcctc gcgtacccga
     6001 ttatccatcg gtggatggag cgactcgtta atcgcttcca tgcgccgcag taacaattgc
     6061 tcaagcagat ttatcgccag cagctccgaa tagcgccctt ccccttgccc ggcgttaatg
     6121 atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt catccgggcg aaagaacccc
     6181 gtattggcaa atattgacgg ccagttaagc cattcatgcc agtaggcgcg cggacgaaag
     6241 taaacccact ggtgatacca ttcgcgagcc tccggatgac gaccgtagtg atgaatctct
     6301 cctggcggga acagcaaaat atcacccggt cggcaaacaa attctcgtcc ctgatttttc
     6361 accaccccct gaccgcgaat ggtgagattg agaatataac ctttcattcc cagcggtcgg
     6421 tcgataaaaa aatcgagata accgttggcc tcaatcggcg ttaaacccgc caccagatgg
     6481 gcattaaacg agtatcccgg cagcagggga tcattttgcg cttcagccat acttttcata
     6541 ctcccgccat tcagag
//
