LOCUS       Exported                5070 bp DNA     circular SYN 15-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 5070)
  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 5070)
  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..5070
                     /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..374
                     /label=pegRNA_RT+PBS
     protein_bind    375..394
                     /label=Csy4 site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     misc_feature    395..414
                     /label=nick-sgRNA_spacer
     misc_RNA        415..490
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    542..659
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        708..1218
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     CDS             1227..1946
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1272..1293)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1880..1901
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2069..2190
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2502..3090)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3261..4121)
                     /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     3884..3903
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4122..4226)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4253..4708
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4340..4359)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4550..4571
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4839..4887
                     /note="synthetic polyadenylation signal"
     misc_feature    4901..4992
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4941..4960
                     /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 ccaccctgtg ctgagtgtat gtttcagagc tagaaatagc aagttgaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctctc tccctccaag
      361 acactcagca cagggttcac tgccgtatag gcagtagctt cctatccaat gccagtttta
      421 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      481 gagtcggtgc tttttttaaa gaattctcga cctcgagaca aatggcagta ttcatccaca
      541 attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
      601 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc
      661 gggtttatta cagggacagc agagatccac tttggccgcg gctcgagggg gttggggttg
      721 cgccttttcc aaggcagccc tgggtttgcg cagggacgcg gctgctctgg gcgtggttcc
      781 gggaaacgca gcggcgccga ccctgggact cgcacattct tcacgtccgt tcgcagcgtc
      841 acccggatct tcgccgctac ccttgtgggc cccccggcga cgcttcctgc tccgccccta
      901 agtcgggaag gttccttgcg gttcgcggcg tgccggacgt gacaaacgga agccgcacgt
      961 ctcactagta ccctcgcaga cggacagcgc cagggagcaa tggcagcgcg ccgaccgcga
     1021 tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg agagcagcgg ccgggaaggg
     1081 gcggtgcggg aggcggggtg tggggcggta gtgtgggccc tgttcctgcc cgcgcggtgt
     1141 tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg gctccctcgt tgaccgaatc
     1201 accgacctct ctccccaggg ggatccatgg tgagcaaggg cgaggagctg ttcaccgggg
     1261 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1321 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1381 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1441 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1501 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1561 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1621 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1681 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     1741 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1801 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     1861 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1921 tcggcatgga cgagctgtac aagtaaagcg gccgcgactc tagatcataa tcagccatac
     1981 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     2041 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     2101 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     2161 tggtttgtcc aaactcatca atgtatctta gtcgaccgat gcccttgaga gccttcaacc
     2221 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct
     2281 tctttatcat gcaactcgta ggacaggtgc cggcagcgct cttccgcttc ctcgctcact
     2341 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     2401 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     2461 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     2521 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     2581 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     2641 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     2701 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     2761 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     2821 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     2881 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     2941 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3001 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3061 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3121 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3181 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3241 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3301 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3361 gagggcttac catctggccc cagtgctgca atgataccgc gggacccacg ctcaccggct
     3421 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3481 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3541 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3601 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3661 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3721 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     3781 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3841 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3901 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3961 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4021 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4081 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4141 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4201 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc
     4261 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4321 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4381 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4441 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     4501 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4561 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     4621 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4681 tttaacaaaa tattaacgct tacaatttgc cattcgccat tcaggctgcg caactgttgg
     4741 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc ccaagctacc atgataagta
     4801 agtaatatta aggtacggga ggtacttgga gcggccgcaa taaaatatct ttattttcat
     4861 tacatctgtg tgttggtttt ttgtgtgaat cgatagtact aacatacgct ctccatcaaa
     4921 acaaaacgaa acaaaacaaa ctagcaaaat aggctgtccc cagtgcaagt gcaggtgcca
     4981 gaacatttct ctatcgatag gtaccgatta gtgaacggat ctcgacggta tcgatcacga
     5041 gactagcctc gagcggccgc ccccttcacc
//
