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基本信息

質粒圖譜和多克隆位點信息

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載體描述

pGAG-IRES-GFP在pGAG-GFP載體的基礎上改造而來,雙順反子載體；熒光報告載體。

CAG啟動子是人工構建的組合啟動子，由巨細胞病毒（the cytomegalovirus，CMV)早期增強子（early enhancer element）和雞β-肌動蛋白（chicken beta-actin）啟動子組成，用于驅動基因在哺乳動物載體的高水平表達。真核表達的啟動子CAG(CMV/actin/Globin)，它減少了單純CMV啟動子在多種細胞中容易被甲基化從而被關閉表達的尷尬。

pGAG-IRES-GFP contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between the MCS and the enhanced green fluorescent protein (EGFP) coding region. This permits both the gene of interest (cloned into the MCS) and the EGFP gene to be translated from a single bicistronic mRNA.

載體序列

LOCUS       Exported                6183 bp ds-DNA     circular SYN 21-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    .

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 6183)

  AUTHORS   BT-Lab

  TITLE     Direct Submission

  JOURNAL   Exported Thursday, Mar 21, 2019 from SnapGene Viewer 4.3.4

FEATURES             Location/Qualifiers

     source          1..6183

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     promoter        4..1718

                     /label=CAG promoter

                     /note="CAG promoter is a combination of artificially

                     constructed promoter, which is composed of the early

                     enhancer element of cytomegalovirus (CMV) and chicken

                     beta-actin promoter, and is used for the high level

                     expression of driver gene in mammalian vector."

     enhancer        4..383

                     /label=CMV enhancer

                     /note="human cytomegalovirus immediate early enhancer"

     promoter        386..661

                     /label=chicken beta-actin promoter

     intron          662..1718

                     /label=chimeric intron

                     /note="chimera between introns from chicken beta-actin and

                     rabbit beta-globin"

     primer_bind     1682..1701

                     /label=pCAG-F

     misc_feature    1729..1814

                     /label=MCS

                     /note="multiple cloning site"

     misc_feature    1816..2402

                     /label=IRES2

                     /note="internal ribosome entry site (IRES) of the

                     encephalomyocarditis virus (EMCV)"

     primer_bind     complement(1911..1930)

                     /label=IRES-R

     CDS             2403..3122

                     /codon_start=1

                     /product="enhanced GFP"

                     /label=EGFP

                     /note="mammalian codon-optimized"

                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL

                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD

                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK

                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL

                     EFVTAAGITLGMDELYK"

     polyA_site      3271..3326

                     /label=beta-globin poly(A) signal

                     /note="rabbit beta-globin polyadenylation signal"

     primer_bind     complement(3686..3702)

                     /label=M13 rev

                     /note="common sequencing primer, one of multiple similar

                     variants"

     protein_bind    3710..3726

                     /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(3734..3764)

                     /label=lac promoter

                     /note="promoter for the E. coli lac operon"

     protein_bind    3779..3800

                     /label=CAP binding site

                     /bound_moiety="E. coli catabolite activator protein"

                     /note="CAP binding activates transcription in the presence

                     of cAMP."

     promoter        3859..4054

                     /label=SV40 promoter

                     /note="SV40 early promoter"

     rep_origin      3905..4040

                     /label=SV40 ori

                     /note="SV40 origin of replication"

     polyA_signal    4060..4194

                     /label=SV40 poly(A) signal

                     /note="SV40 polyadenylation signal"

     rep_origin      complement(4433..5021)

                     /direction=LEFT

                     /label=ori

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

                     replication"

     CDS             complement(5192..6052)

                     /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(6053..6157)

                     /gene="bla"

                     /label=AmpR promoter

ORIGIN

        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata

       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc

      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag

      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac

      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg

      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg

      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc

      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca

      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg

      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt

      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc

      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc

      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc

      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag

      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg

      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg

      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc

     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg

     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc

     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc

     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg

     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc

     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag

     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc

     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc

     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg

     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc

     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg

     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattcgg cgcgccatcg

     1741 atacgcgtca attgcggccg cctcgagctc aagcttctag ttctgcagtc gacggtaccg

     1801 cgggcccggg atccgcccct ctccctcccc cccccctaac gttactggcc gaagccgctt

     1861 ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg

     1921 caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc

     1981 ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga

     2041 agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc

     2101 tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc

     2161 acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc

     2221 aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga

     2281 tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg

     2341 ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa

     2401 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg

     2461 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct

     2521 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca

     2581 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga

     2641 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct

     2701 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc

     2761 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc

     2821 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga

     2881 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg

     2941 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc

     3001 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg

     3061 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt

     3121 aagaattcac tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg

     3181 ccctggctca caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca

     3241 tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag

     3301 tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa

     3361 aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca

     3421 tgaacaaagg tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc

     3481 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt

     3541 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct

     3601 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatgaagat ccctcgacct

     3661 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc

     3721 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat

     3781 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc

     3841 tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc

     3901 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat

     3961 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg

     4021 aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat

     4081 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg

     4141 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc

     4201 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt

     4261 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact

     4321 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag

     4381 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata

     4441 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc

     4501 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg

     4561 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc

     4621 tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg

     4681 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc

     4741 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga

     4801 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg

     4861 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa

     4921 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg

     4981 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt

     5041 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat

     5101 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct

     5161 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta

     5221 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa

     5281 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac

     5341 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa

     5401 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag

     5461 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg

     5521 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag

     5581 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg

     5641 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc

     5701 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat

     5761 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata

     5821 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa

     5881 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca

     5941 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc

     6001 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc

     6061 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg

     6121 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac

     6181 ctg

//