LOCUS       Exported                4957 bp ds-DNA     circular SYN 18-AUG-2020
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 4957)
  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 4957)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Aug 21, 2020 from SnapGene Viewer 5.1.5
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Commun.
            2018 Jun 14"; volume: "9(1):2338. doi"; pages: " 
            10.1038/s41467-018-04768-7"
FEATURES             Location/Qualifiers
     source          1..4957
                     /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_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    429..546
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     557..576
                     /label=newallGFP-F
     primer_bind     complement(559..578)
                     /label=allpeg-R
     primer_bind     564..583
                     /label=allGFP-F
     promoter        595..1105
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1024..1042
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1114..1833
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1159..1180)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1420..1439)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1767..1788
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1956..2077
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1993..2012)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2047..2066
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2218..2235)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2389..2977)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2469..2488)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3148..4008)
                     /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     3771..3790
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4009..4113)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4140..4595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4227..4246)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4437..4458
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4726..4774
                     /note="synthetic polyadenylation signal"
     misc_feature    4788..4879
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4828..4847
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     complement(4900..4919)
                     /label=allGFP-R
     primer_bind     4904..4923
                     /label=allpeg-F
     primer_bind     complement(4907..4926)
                     /label=newallGFP-R
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 cccggcatcc acaaatcacc gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcgctc ccctctccag
      361 gagatttgtg gatgccgttt ttttaaagaa ttctcgacct cgagacaaat ggcagtattc
      421 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
      481 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
      541 aattttcggg tttattacag ggacagcaga gatccacttt ggccgcggct cgagggggtt
      601 ggggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct gctctgggcg
      661 tggttccggg aaacgcagcg gcgccgaccc tgggactcgc acattcttca cgtccgttcg
      721 cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc
      781 gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc
      841 cgcacgtctc actagtaccc tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg
      901 accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga gcagcggccg
      961 ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc
     1021 gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct ccctcgttga
     1081 ccgaatcacc gacctctctc cccaggggga tccatggtga gcaagggcga ggagctgttc
     1141 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1201 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1261 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     1321 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1381 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1441 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1501 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1561 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     1621 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     1681 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     1741 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1801 atcactctcg gcatggacga gctgtacaag taaagcggcc gcgactctag atcataatca
     1861 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     1921 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     1981 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2041 ctagttgtgg tttgtccaaa ctcatcaatg tatcttagtc gaccgatgcc cttgagagcc
     2101 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
     2161 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctt ccgcttcctc
     2221 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     2281 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     2341 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2401 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2461 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2521 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2581 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2641 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2701 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2761 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2821 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2881 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2941 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3001 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3061 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3121 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3181 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3241 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcggg acccacgctc
     3301 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     3361 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     3421 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     3481 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     3541 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     3601 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     3661 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     3721 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     3781 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     3841 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     3901 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     3961 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4021 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4081 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     4141 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4201 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4261 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4321 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     4381 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4441 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4501 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4561 cgcgaatttt aacaaaatat taacgcttac aatttgccat tcgccattca ggctgcgcaa
     4621 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagccca agctaccatg
     4681 ataagtaagt aatattaagg tacgggaggt acttggagcg gccgcaataa aatatcttta
     4741 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc
     4801 catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca
     4861 ggtgccagaa catttctcta tcgataggta ccgattagtg aacggatctc gacggtatcg
     4921 atcacgagac tagcctcgag cggccgcccc cttcacc
//
