LOCUS       Exported                5069 bp DNA     circular SYN 14-JUN-2021
DEFINITION  For expression of sgRNA from the U6 promoter with EGFP expression 
            simultaneously.
ACCESSION   .
VERSION     .
KEYWORDS    pGL3-U6-sgRNA-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5069)
  AUTHORS   Liu Z, Lu Z, Yang G, Huang S, Li G, Feng S, Liu Y, Li J, Yu W, Zhang
            Y, Chen J, Sun Q, Huang X
  TITLE     Efficient generation of mouse models of human diseases via ABE- and 
            BE-mediated base editing.
  JOURNAL   Nat Commun. 2018 Jun 14;9(1):2338. doi: 10.1038/s41467-018-04768-7.
  PUBMED    29904106
REFERENCE   2  (bases 1 to 5069)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 16, 2021 from SnapGene Viewer 5.3.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2018-06-14"; volume: "9(1):2338. doi"; pages: " 
            10.1038/s41467-018-04768-7"
FEATURES             Location/Qualifiers
     source          1..5069
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    251..270
                     /label=pegRNA_spacer
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    347..373
                     /label=pegRNA_RT+PBS
     protein_bind    374..393
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     misc_feature    394..413
                     /label=nick-sgRNA_spacer
     misc_RNA        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    541..658
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        707..1217
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     CDS             1226..1945
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1271..1292)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1879..1900
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2068..2189
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2501..3089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3260..4120)
                     /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"
     primer_bind     3883..3902
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4121..4225)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4252..4707
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4339..4358)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4549..4570
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4838..4886
                     /note="synthetic polyadenylation signal"
     misc_feature    4900..4991
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4940..4959
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ctcaggtaat ccacctgcct gtttcagagc tagaaatagc aagttgaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcttgg gaggccgagt
      361 caggtggatt accgttcact gccgtatagg cagattttat aggccaggcg tgggttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttaaag aattctcgac ctcgagacaa atggcagtat tcatccacaa
      541 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
      601 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
      661 ggtttattac agggacagca gagatccact ttggccgcgg ctcgaggggg ttggggttgc
      721 gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg cgtggttccg
      781 ggaaacgcag cggcgccgac cctgggactc gcacattctt cacgtccgtt cgcagcgtca
      841 cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa
      901 gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc
      961 tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat
     1021 gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc cgggaagggg
     1081 cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt
     1141 ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca
     1201 ccgacctctc tccccagggg gatccatggt gagcaagggc gaggagctgt tcaccggggt
     1261 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     1321 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     1381 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     1441 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     1501 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     1561 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1621 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     1681 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     1741 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1801 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     1861 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     1921 cggcatggac gagctgtaca agtaaagcgg ccgcgactct agatcataat cagccatacc
     1981 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     2041 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     2101 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2161 ggtttgtcca aactcatcaa tgtatcttag tcgaccgatg cccttgagag ccttcaaccc
     2221 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt
     2281 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg
     2341 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2401 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2461 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2521 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2581 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2641 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2701 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2761 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2821 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2881 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     2941 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3001 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3061 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3121 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3181 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3241 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3301 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3361 agggcttacc atctggcccc agtgctgcaa tgataccgcg ggacccacgc tcaccggctc
     3421 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3481 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3541 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3601 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3661 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3721 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     3781 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     3841 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     3901 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     3961 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4021 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4081 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4141 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4201 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct
     4261 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     4321 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     4381 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4441 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     4501 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4561 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     4621 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4681 ttaacaaaat attaacgctt acaatttgcc attcgccatt caggctgcgc aactgttggg
     4741 aagggcgatc ggtgcgggcc tcttcgctat tacgccagcc caagctacca tgataagtaa
     4801 gtaatattaa ggtacgggag gtacttggag cggccgcaat aaaatatctt tattttcatt
     4861 acatctgtgt gttggttttt tgtgtgaatc gatagtacta acatacgctc tccatcaaaa
     4921 caaaacgaaa caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg caggtgccag
     4981 aacatttctc tatcgatagg taccgattag tgaacggatc tcgacggtat cgatcacgag
     5041 actagcctcg agcggccgcc cccttcacc
//
