pET- 24a Plasmid

PVT0327  2ug

pET- 24a Plasmid Information

Promoter: T7/lac

Copier: ColE1 ori, F1 ori

Terminator: T7 Terminator

Plasmid Classification: E. coli Vector; PET Series Expression Plasmid

Plasmid size: 5310 BP

Plasmid label: N-T7, C-6 *His

Prokaryotic resistance: Kanamycin/ Kan

Cloning strain: DH5alpha

Culture conditions: 37 ℃, aerobic, LB

Expression host: BL21 (DE3)

Induction: IPTG or lactose and its analogues

5'Sequencing Primers: T7: TAATACGACTCACTATAGG

3'Sequencing primers: T7-ter: TGCTAGTTATTGCTCAGCGG

pET- 24a Plasmid Description

The pET-24a-d(+) vectors carry an N-terminal T7•Tag® sequence plus an optional C-terminal His•Tag® sequence. These vectors differ from pET-21a-d(+) only by their selectable marker (kanamycin vs. ampicillin resistance). Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circular map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNA that corresponds to the coding strand. Therefore, singlestranded sequencing should be performed using the T7 terminator primer .

pET- 24a Plasmid Sites

pET- 24a Plasmid Sequence

LOCUS       Exported                5310 bp ds-DNA     circular SYN 31-7-2015
DEFINITION  synthetic circular DNA
KEYWORDS    Untitled 4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5310)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 2015-7-31  
FEATURES             Location/Qualifiers
     source          1..5310
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          247..269
                     /organism="Enterobacteria phage T7"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:10760"
     terminator      26..73
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(207..239)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /note="T7 tag (gene 10 leader)"
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     RBS             247..269
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    284..308
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(309..327)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        636..713
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             714..1796
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     CDS             2605..2796
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    2898..3040
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3226..3814)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             3936..4751
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin in bacteria or G418
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(4844..5299)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccgcgaccc atttgctgtc caccagtcat gctagccata
      241 tgtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc
      301 acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc
      361 cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
      421 cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg
      481 cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc
      541 accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat
      601 gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct
      661 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
      721 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
      781 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
      841 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
      901 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
      961 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     1021 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     1081 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     1141 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     1201 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     1261 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     1321 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     1381 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     1441 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     1501 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     1561 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     1621 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     1681 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     1741 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     1801 aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg
     1861 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
     1921 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc
     1981 attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta
     2041 ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc
     2101 ggcgagaagc aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc
     2161 ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca
     2221 ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca
     2281 acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc
     2341 tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct
     2401 acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc
     2461 atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca
     2521 gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga
     2581 aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc
     2641 cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat
     2701 gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc
     2761 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     2821 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     2881 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
     2941 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatata tgcggtgtga
     3001 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct
     3061 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3121 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3181 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3241 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3301 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3361 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3421 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3481 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3541 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3601 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3661 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3721 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3781 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3841 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac
     3901 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
     3961 cttgctctag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     4021 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg
     4081 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     4141 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     4201 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg
     4261 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     4321 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     4381 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     4441 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
     4501 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     4561 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     4621 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     4681 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     4741 agtttttcta agaattaatt catgagcgga tacatatttg aatgtattta gaaaaataaa
     4801 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgaaattgt aaacgttaat
     4861 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     4921 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     4981 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5041 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5101 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     5161 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     5221 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     5281 gcgccgctac agggcgcgtc ccattcgcca
//

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