LOCUS       Exported                5519 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  FX cloning Lactococcus lactis expression vector (VBEx) with nisin 
            promoter and C-terminal 3C protease cleavage site and 10x His tags.
ACCESSION   .
VERSION     .
KEYWORDS    pREXC3H
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5519)
  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 5519)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5519
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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(1405..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"
     CDS             complement(1627..1647)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1674..1697
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1698..1727
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     terminator      2183..2210
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2229..2320
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2321..3181
                     /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(2539..2558)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3352..3940
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3841..3860
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4094..4111
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4192..4211)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gaagagcgac 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 tggatctacc tttctcttct tttttgggga tccactagtt cagctcttct gcattagaag
     1681 ttttgtttca aggtccacac catcatcatc accatcatca tcatcattaa taatctagag
     1741 agctcaagct ttctttgaac caaaattaga aaaccaaggc ttgaaacgtt caattgaaat
     1801 ggcaattaaa caaattacag cacgtgttgc tttgattgat agccaaaaag cagcagttga
     1861 taaagcaatt actgatattg ctgaaaaatt gtaatttata aataaaaatc accttttaga
     1921 ggtggttttt ttatttataa attattcgtt tgatttcgct ttcgatagaa caatcaaagc
     1981 gagaataagg aagataaatc ccataagggc gggagcagaa tgtccgagac taatgtaaat
     2041 ttgtccacca attaaaggac cgataacgcg agcttctcga gatggccact gaggccggga
     2101 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
     2161 actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta
     2221 caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2281 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     2341 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     2401 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     2461 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     2521 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     2581 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     2641 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     2701 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     2761 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2821 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     2881 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2941 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3001 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     3061 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     3121 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     3181 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     3241 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3301 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3361 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3421 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3481 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3541 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3601 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3661 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3721 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3781 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3841 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3901 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3961 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4021 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4081 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gtgcccctga tgcggtattt
     4141 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     4201 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     4261 gccggacggg ccatgtcgac ggcaatagtt acccttatta tcaagataag aaagaaaagg
     4321 atttttcgct acgctcaaat cctttaaaaa aacacaaaag accacatttt ttaatgtggt
     4381 ctttattctt caactaaagc acccattagt tcaacaaacg aaaattggat aaagtgggat
     4441 atttttaaaa tatatattta tgttacagta atattgactt ttaaaaaagg attgattcta
     4501 atgaagaaag cagacaagta agcctcctaa attcacttta gataaaaatt taggaggcat
     4561 atcaaatgaa ctttaataaa attgatttag acaattggaa gagaaaagag atatttaatc
     4621 attatttgaa ccaacaaacg acttttagta taaccacaga aattgatatt agtgttttat
     4681 accgaaacat aaaacaagaa ggatataaat tttaccctgc atttattttc ttagtgacaa
     4741 gggtgataaa ctcaaataca gcttttagaa ctggttacaa tagcgacgga gagttaggtt
     4801 attgggataa gttagagcca ctttatacaa tttttgatgg tgtatctaaa acattctctg
     4861 gtatttggac tcctgtaaag aatgacttca aagagtttta tgatttatac ctttctgatg
     4921 tagagaaata taatggttcg gggaaattgt ttcccaaaac acctatacct gaaaatgctt
     4981 tttctctttc tattattcct tggacttcat ttactgggtt taacttaaat atcaataata
     5041 atagtaatta ccttctaccc attattacag caggaaaatt cattaataaa ggtaattcaa
     5101 tatatttacc gctatcttta caggtacatc attctgtttg tgatggttat catgctggat
     5161 tgtttatgaa ctctattcag gaattgtcag ataggcctaa tgactggctt ttataatatg
     5221 agataatgcc gactgtactt tttacagtcg gttttctaat gtcactaacc tgccccgtta
     5281 gttgaagaag gtttttatat tacagctcca gatctagtct tataactata ctgacaatag
     5341 aaacattaac aaatctaaaa cagtcttaat tctatcttga gaaagtattg gtaataatat
     5401 tattgtcgat aacgcgagca taataaacgg ctctgattaa attctgaagt ttgttagata
     5461 caatgatttc gttcgaagga actacaaaat aaattataag gaggcactca ccatgagtt
//
