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質粒圖譜和多克隆位點信息

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載體描述

pQE-40 pQE vectors and constructs can be maintained in any E. coli strain that is ampicillin-sensitive and carries the pREP4 repressor plasmid, or harbors the lacIq gene on the F-factor episome. 配套表達菌株為M15[pREP4]感受態細胞。M15[pREP4]菌株攜帶有阻抑質粒pREP4,該質粒高水平的表達lac抑制子，在IPTG誘導前反式抑制蛋白表達。通過卡那霉素抗性篩選可以保持pREP4質粒在菌株中的穩定性。

DHFR是指二氫葉酸還原酶，CHO-DHFR細胞是二氫葉酸還原酶雙倍體基因缺失型CHO細胞，在缺乏DHFR的情況下無法生長，可以轉染表達DHFR的質粒進入該細胞系，這樣，轉染成功的細胞才能存活，其他細胞死亡，可以作為穩定細胞系構建的篩選系統。

載體序列

LOCUS       Exported                4031 bp ds-DNA     circular SYN 16-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    .

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 4031)

  AUTHORS   .

  TITLE     Direct Submission

  JOURNAL   Exported Saturday, Mar 16, 2019 from SnapGene Viewer 4.3.4

FEATURES             Location/Qualifiers

     source          1..4031

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     promoter        10..54

                     /label=T5 promoter

                     /note="bacteriophage T5 promoter for E. coli RNA

                     polymerase, with embedded lac operator"

     protein_bind    30..46

                     /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)."

     protein_bind    62..78

                     /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)."

     primer_bind     67..88

                     /label=pQE30-F

     RBS             97..108

                     /note="strong bacterial ribosome binding site (Elowitz and

                     Leibler, 2000)"

     CDS             127..144

                     /codon_start=1

                     /product="6xHis affinity tag"

                     /label=6xHis

                     /translation="HHHHHH"

     CDS             157..714

                     /codon_start=1

                     /gene="Mus musculus Dhfr"

                     /product="mouse dihydrofolate reductase"

                     /label=DHFR

                     /translation="MVRPLNSIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVE

                     GKQNLVIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQP

                     ELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEY

                     PGVLSEVQEEKGIKYKFEVYEKK"

     misc_feature    721..765

                     /label=MCS

     terminator      778..872

                     /label=lambda t0 terminator

                     /note="transcription terminator from phage lambda"

     primer_bind     complement(792..812)

                     /label=pQE30-R

     CDS             916..1575

                     /codon_start=1

                     /gene="cat"

                     /product="chloramphenicol acetyltransferase"

                     /label=CmR

                     /note="confers resistance to chloramphenicol"

                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL

                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS

                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM

                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"

     terminator      1640..1726

                     /gene="Escherichia coli rrnB"

                     /label=rrnB T1 terminator

                     /note="transcription terminator T1 from the E. coli rrnB

                     gene"

     misc_feature    1881..2021

                     /label=bom

                     /note="basis of mobility region from pBR322"

     rep_origin      complement(2207..2795)

                     /direction=LEFT

                     /label=ori

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

                     replication"

     CDS             complement(2966..3826)

                     /codon_start=1

                     /gene="bla"

                     /product="beta-lactamase"

                     /label=AmpR

                     /note="confers resistance to ampicillin, carbenicillin, and

                     related antibiotics"

                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI

                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS

                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW

                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA

                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS

                     LIKHW"

     promoter        complement(3827..3931)

                     /gene="bla"

                     /label=AmpR promoter

ORIGIN

        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca

       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga

      121 ggatcgcatc accatcacca tcacggatcc ggcatcatgg ttcgaccatt gaactcgatc

      181 gtcgccgtgt cccaaaatat ggggattggc aagaacggag acctaccctg gcctccgctc

      241 aggaacgagt tcaagtactt ccaaagaatg accacaacct cttcagtgga aggtaaacag

      301 aatctggtga ttatgggtag gaaaacctgg ttctccattc ctgagaagaa tcgaccttta

      361 aaggacagaa ttaatatagt tctcagtaga gaactcaaag aaccaccacg aggagctcat

      421 tttcttgcca aaagtttgga tgatgcctta agacttattg aacaaccgga attggcaagt

      481 aaagtagaca tggtttggat agtcggaggc agttctgttt accaggaagc catgaatcaa

      541 ccaggccacc ttagactctt tgtgacaagg atcatgcagg aatttgaaag tgacacgttt

      601 ttcccagaaa ttgatttggg gaaatataaa cttctcccag aatacccagg cgtcctctct

      661 gaggtccagg aggaaaaagg catcaagtat aagtttgaag tctacgagaa gaaaggttcc

      721 agatctgcat gcggtacccc gggtcgacct gcagccaagc ttaattagct gagcttggac

      781 tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg

      841 gttgccgccg ggcgtttttt attggtgaga atccaagcta gcttggcgag attttcagga

      901 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa

      961 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag

     1021 accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt

     1081 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga atttcgtatg

     1141 gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc

     1201 catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag

     1261 tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct

     1321 aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt

     1381 tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa

     1441 tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt

     1501 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg

     1561 cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc tggggtaatg

     1621 actctctagc ttgaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg

     1681 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cctctagagc

     1741 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg

     1801 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg

     1861 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat

     1921 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg

     1981 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc

     2041 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg

     2101 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag

     2161 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc

     2221 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag

     2281 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga

     2341 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc

     2401 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg

     2461 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt

     2521 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca

     2581 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca

     2641 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag

     2701 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca

     2761 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg

     2821 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa

     2881 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta

     2941 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag

     3001 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga

     3061 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac

     3121 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc

     3181 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta

     3241 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac

     3301 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat

     3361 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa

     3421 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg

     3481 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag

     3541 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc

     3601 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct

     3661 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat

     3721 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg

     3781 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc

     3841 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta

     3901 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg

     3961 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct

     4021 ttcgtcttca c

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