• 规格：20μl质粒
• 原核抗性：氨苄青霉素Amp
• 筛选标记：嘌呤霉素Puro
• 克隆菌株：大肠杆菌DH5α
• 备注：序列和Addgene有差异
• 质粒简介：            pGL3-U6-sgRNA-PGK-puromycin是一个哺乳细胞基因编辑质粒，U6启动sgRNA的转录，可用BsaI酶切连入sgRNA序列，pGK启动嘌呤霉素Puro抗性基因的表达。
• 质粒图谱1： png/9-1P605093255Z3.png

质粒序列： 点击查看序列

LOCUS       Exported                4951 bp ds-DNA    circular SYN 17-七月-2017

KEYWORDS    pGL3-U6-sgRNA-PGK-puro

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 4951)

  AUTHORS   MiaoLingBio

  TITLE     Direct Submission

FEATURES             Location/Qualifiers

     source          1..4951

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     promoter        72..312

                     /label="U6 Promoter"

                     /note="U6 promoter"

                     /note="RNA polymerase III promoter for human U6 snRNA"

     misc_RNA        343..418

                     /note="gRNA scaffold"

                     /note="guide RNA scaffold for the CRISPR/Cas9 system"

     misc_feature    470..587

                     /note="cPPT/CTS"

                     /note="central polypurine tract and central termination 

                     sequence of HIV-1"

     promoter        636..1146

                     /note="hPGK promoter"

                     /note="human phosphoglycerate kinase 1 promoter"

     CDS             1168..1767

                     /codon_start=1

                     /gene="pac from Streptomyces alboniger"

                     /product="puromycin N-acetyltransferase"

                     /note="PuroR"

                     /note="confers resistance to puromycin"

                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER

                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA

                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS

                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"

     polyA_signal    1914..2035

                     /note="SV40 poly(A) signal"

                     /note="SV40 polyadenylation signal"

     rep_origin      complement(2454..3042)

                     /direction=LEFT

                     /note="ori"

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

                     replication"

     CDS             complement(3213..4073)

                     /codon_start=1

                     /gene="bla"

                     /product="beta-lactamase"

                     /note="AmpR"

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

                     related antibiotics"

                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI

                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS

                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW

                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA

                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS

                     LIKHW"

     promoter        complement(4074..4178)

                     /gene="bla"

                     /note="AmpR promoter"

     rep_origin      4205..4660

                     /direction=RIGHT

                     /note="f1 ori"

                     /note="f1 bacteriophage origin of replication; arrow 

                     indicates direction of (+) strand synthesis"

     polyA_signal    4791..4839

                     /note="synthetic polyadenylation signal"

     misc_feature    4853..4944

                     /note="pause site"

                     /note="RNA polymerase II transcriptional pause signal from 

                     the human alpha-2 globin gene"

ORIGIN

        1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg

       61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg

      121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata

      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa

      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct

      301 tgtggaaagg acgaaacacc gtgagaccga gagagggtct cagttttaga gctagaaata

      361 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt

      421 tttttaaaga attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa

      481 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca

      541 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca

      601 gggacagcag agatccactt tggccgcggc tcgagggggt tggggttgcg ccttttccaa

      661 ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc

      721 ggcgccgacc ctgggactcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc

      781 gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt

      841 tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc

      901 ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc

      961 aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag

     1021 gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc

     1081 aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct

     1141 ccccaggggg atccaccgga gcttaccatg accgagtaca agcccacggt gcgcctcgcc

     1201 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc

     1261 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa

     1321 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc

     1381 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc

     1441 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc

     1501 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg

     1561 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc

     1621 gagcgcgccg gggtgcccgc cttcctggaa acctccgcgc cccgcaacct ccccttctac

     1681 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg

     1741 tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa

     1801 aggagcgcac gaccccatgc atcggtacct ttaagaccaa tgacttacaa ggcagctgta

     1861 gatcttagcc actttctaga gtcggggcgg ccggccgctt cgagcagaca tgataagata

     1921 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga

     1981 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa

     2041 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag

     2101 caagtaaaac ctctacaaat gtggtaaaat cgataaggat ccgtcgaccg atgcccttga

     2161 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac

     2221 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctcttccgct

     2281 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac

     2341 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga

     2401 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat

     2461 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac

     2521 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct

     2581 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg

     2641 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg

     2701 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt

     2761 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg

     2821 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac

     2881 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga

     2941 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt

     3001 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt

     3061 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga

     3121 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc

     3181 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct

     3241 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata

     3301 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgggaccca

     3361 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga

     3421 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga

     3481 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg

     3541 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga

     3601 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt

     3661 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct

     3721 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca

     3781 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat

     3841 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga

     3901 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc

     3961 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg

     4021 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc

     4081 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt

     4141 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca

     4201 cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg

     4261 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc

     4321 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga

     4381 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt

     4441 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat

     4501 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat

     4561 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa

     4621 tttaacgcga attttaacaa aatattaacg tttacaattt cccattcgcc attcaggctg

     4681 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gcccaagcta

     4741 ccatgataag taagtaatat taaggtacgg gaggtacttg gagcggccgc aataaaatat

     4801 ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta ctaacatacg

     4861 ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc cccagtgcaa

     4921 gtgcaggtgc cagaacattt ctctatcgat a

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