LOCUS       sequence-67105-e        5494 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FX cloning Lactococcus lactis expression vector (VBEx) with nisin 
            promoter and N-terminal 10x His tag and 3C protease cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5494)
  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 5494)
  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: pREXNH3
FEATURES             Location/Qualifiers
     source          1..5494
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(45..350)
                     /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(147..166)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(692..1351)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1266..1285
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1405..1411,1412..1429,1430..1435))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(1460..1584)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1603..1623)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      2104..2131
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2150..2241
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2242..2310,2311..3102)
                     /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(2460..2479)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3273..3861
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3762..3781
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4015..4032
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4113..4132)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             5437..5466
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5467..5490
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 gaagagcgag ctgcagactg gctgtgtata agggagcctg acatttatat tccccagaac
       61 atcaggttaa tggcgttttt gatgtcattt tcgcggtggc tgagatcagc cacttcttcc
      121 ccgataacgg agaccggcac actggccata tcggtggtca tcatgcgcca gctttcatcc
      181 ccgatatgca ccaccgggta aagttcacgg gagactttat ctgacagcag acgtgcactg
      241 gccaggggga tcaccatccg tcgcccgggc gtgtcaataa tatcactctg tacatccaca
      301 aacagacgat aacggctctc tcttttatag gtgtaaacct taaactgcat ttcaccagcc
      361 cctgttctcg tcagcaaaag agccgttcat ttcaataaac cgggcgacct cagccatccc
      421 ttcctgattt tccgctttcc agcgttcggc acgcagacga cgggcttcat tctgcatggt
      481 tgtgcttacc agaccggaga tattgacatc atatatgcct tgagcaactg atagctgtcg
      541 ctgtcaactg tcactgtaat acgctgcttc atagcatacc tctttttgac atacttcggg
      601 tatacatatc agtatatatt cttataccgc aaaaatcagc gcgcaaatac gcatactgtt
      661 atctggcttt tagtaagccg gatccacgcg tttacgcccc gccctgccac tcatcgcagt
      721 actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg gcatgatgaa
      781 cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatggtga
      841 aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca
      901 cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg aaataggcca
      961 ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt
     1021 cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac
     1081 aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata cggaattccg
     1141 gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat
     1201 ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac
     1261 attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa
     1321 cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg
     1381 acggatccta actcaaaatc cacacattat acgagccgga agcataaagt gtaaagcctg
     1441 gggtgcctaa tgcggccgcc atagtgactg gatatgttgt gttttacagt attatgtagt
     1501 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
     1561 ttcagctttt ttgtacaaac ttgtgatatc gaattcgcta gctacctttc tcttcttttt
     1621 tggggatcca ctagttcagc tcttctgcat aataactaga gagctcaagc tttctttgaa
     1681 ccaaaattag aaaaccaagg cttgaaacgt tcaattgaaa tggcaattaa acaaattaca
     1741 gcacgtgttg ctttgattga tagccaaaaa gcagcagttg ataaagcaat tactgatatt
     1801 gctgaaaaat tgtaatttat aaataaaaat caccttttag aggtggtttt tttatttata
     1861 aattattcgt ttgatttcgc tttcgataga acaatcaaag cgagaataag gaagataaat
     1921 cccataaggg cgggagcaga atgtccgaga ctaatgtaaa tttgtccacc aattaaagga
     1981 ccgataacgc gagcttctcg agatggccac tgaggccggg agcggatttg aacgttgcga
     2041 agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta
     2101 agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt
     2161 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2221 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     2281 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     2341 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     2401 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     2461 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
     2521 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     2581 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     2641 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     2701 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     2761 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     2821 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     2881 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     2941 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     3001 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3061 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3121 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     3181 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3241 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3301 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3361 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3421 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3481 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3541 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3601 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3661 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3721 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3781 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3841 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3901 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3961 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4021 tcagtgagcg aggaagcgga agtgcccctg atgcggtatt ttctccttac gcatctgtgc
     4081 ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4141 agccagtata cactccgcta tcgctacgtg actgggtcat ggccggacgg gccatgtcga
     4201 cggcaatagt tacccttatt atcaagataa gaaagaaaag gatttttcgc tacgctcaaa
     4261 tcctttaaaa aaacacaaaa gaccacattt tttaatgtgg tctttattct tcaactaaag
     4321 cacccattag ttcaacaaac gaaaattgga taaagtggga tatttttaaa atatatattt
     4381 atgttacagt aatattgact tttaaaaaag gattgattct aatgaagaaa gcagacaagt
     4441 aagcctccta aattcacttt agataaaaat ttaggaggca tatcaaatga actttaataa
     4501 aattgattta gacaattgga agagaaaaga gatatttaat cattatttga accaacaaac
     4561 gacttttagt ataaccacag aaattgatat tagtgtttta taccgaaaca taaaacaaga
     4621 aggatataaa ttttaccctg catttatttt cttagtgaca agggtgataa actcaaatac
     4681 agcttttaga actggttaca atagcgacgg agagttaggt tattgggata agttagagcc
     4741 actttataca atttttgatg gtgtatctaa aacattctct ggtatttgga ctcctgtaaa
     4801 gaatgacttc aaagagtttt atgatttata cctttctgat gtagagaaat ataatggttc
     4861 ggggaaattg tttcccaaaa cacctatacc tgaaaatgct ttttctcttt ctattattcc
     4921 ttggacttca tttactgggt ttaacttaaa tatcaataat aatagtaatt accttctacc
     4981 cattattaca gcaggaaaat tcattaataa aggtaattca atatatttac cgctatcttt
     5041 acaggtacat cattctgttt gtgatggtta tcatgctgga ttgtttatga actctattca
     5101 ggaattgtca gataggccta atgactggct tttataatat gagataatgc cgactgtact
     5161 ttttacagtc ggttttctaa tgtcactaac ctgccccgtt agttgaagaa ggtttttata
     5221 ttacagctcc agatctagtc ttataactat actgacaata gaaacattaa caaatctaaa
     5281 acagtcttaa ttctatcttg agaaagtatt ggtaataata ttattgtcga taacgcgagc
     5341 ataataaacg gctctgatta aattctgaag tttgttagat acaatgattt cgttcgaagg
     5401 aactacaaaa taaattataa ggaggcactc accatgcacc atcatcatca ccatcatcat
     5461 catcatttag aagttttgtt tcaaggtcca agta
//
