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載體描述

The pET-26b(+) vector (Cat. No. 69862-3) carries an N-terminal pelB signal sequence for potential periplasmic localization, plus optional C-terminal His?Tag? sequence. 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, single-stranded sequencing should be performed using the T7 terminator primer (Cat. No. 69337-3).

載體序列

LOCUS       Exported                5360 bp ds-DNA     circular SYN 14-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    .

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 5360)

  AUTHORS   .

  TITLE     Direct Submission

  JOURNAL   Exported Thursday, Mar 14, 2019 from SnapGene Viewer 4.3.4

FEATURES             Location/Qualifiers

     source          1..5360

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     terminator      26..73

                     /label=T7 terminator

                     /note="transcription terminator for bacteriophage T7 RNA

                     polymerase"

     CDS             complement(140..157)

                     /codon_start=1

                     /product="6xHis affinity tag"

                     /label=6xHis

                     /translation="HHHHHH"

     misc_feature    158..223

                     /label=MCS

     sig_peptide     complement(224..289)

                     /gene="pelB (fragment)"

                     /product="leader peptide for secretion"

                     /label=pelB signal sequence

     RBS             297..319

                     /note="efficient ribosome binding site from bacteriophage

                     T7 gene 10 (Olins and Rangwala, 1989)"

     protein_bind    334..358

                     /label=lac operator

                     /bound_moiety="lac repressor encoded by 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)."

     promoter        complement(359..377)

                     /label=T7 promoter

                     /note="promoter for bacteriophage T7 RNA polymerase"

     promoter        686..763

                     /gene="lacI"

                     /label=lacI promoter

     CDS             764..1846

                     /codon_start=1

                     /gene="lacI"

                     /product="lac repressor"

                     /label=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             2655..2846

                     /codon_start=1

                     /gene="rop"

                     /product="Rop protein, which maintains plasmids at low copy

                     number"

                     /label=rop

                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA

                     DELYRSCLARFGDDGENL"

     misc_feature    2948..3090

                     /label=bom

                     /note="basis of mobility region from pBR322"

     rep_origin      complement(3276..3864)

                     /direction=LEFT

                     /label=ori

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of

                     replication"

     CDS             3986..4801

                     /codon_start=1

                     /gene="aph(3')-Ia"

                     /product="aminoglycoside phosphotransferase"

                     /label=KanR

                     /note="confers resistance to kanamycin in bacteria or G418

                     (Geneticin(R)) in eukaryotes"

                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG

                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK

                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA

                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI

                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"

     rep_origin      complement(4894..5349)

                     /direction=LEFT

                     /label=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 tccgaattaa ttccgatatc catggccatc gccggctggg

      241 cagcgaggag cagcagacca gcagcagcgg tcggcagcag gtatttcata tgtatatctc

      301 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc

      361 tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc

      421 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc

      481 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg

      541 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt

      601 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg

      661 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat

      721 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt

      781 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca

      841 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa

      901 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt

      961 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg

     1021 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc

     1081 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta

     1141 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt

     1201 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg

     1261 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc

     1321 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac

     1381 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt

     1441 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa

     1501 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc

     1561 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt

     1621 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca

     1681 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag

     1741 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt

     1801 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta

     1861 atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca

     1921 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg

     1981 tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg

     2041 aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct

     2101 tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc

     2161 aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga

     2221 ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg

     2281 agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg

     2341 tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta

     2401 tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat

     2461 taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg

     2521 ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta

     2581 tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt

     2641 acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca

     2701 gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag

     2761 acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt

     2821 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc

     2881 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt

     2941 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta

     3001 tgcggcatca gagcagattg tactgagagt gcaccatata tgcggtgtga aataccgcac

     3061 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg

     3121 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg

     3181 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag

     3241 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac

     3301 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga

     3361 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt

     3421 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc

     3481 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc

     3541 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta

     3601 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat

     3661 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca

     3721 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct

     3781 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt

     3841 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct

     3901 cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta

     3961 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag

     4021 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa

     4081 tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt

     4141 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact

     4201 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga

     4261 tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga

     4321 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca

     4381 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc

     4441 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg

     4501 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc

     4561 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat

     4621 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct

     4681 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg

     4741 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta

     4801 agaattaatt catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg

     4861 ttccgcgcac atttccccga aaagtgccac ctgaaattgt aaacgttaat attttgttaa

     4921 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca

     4981 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga

     5041 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc

     5101 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc

     5161 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc

     5221 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg

     5281 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac

     5341 agggcgcgtc ccattcgcca

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