LOCUS       Exported                4959 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 4959)
  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 4959)
  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..4959
                     /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    431..548
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     559..578
                     /label=newallGFP-F
     primer_bind     complement(561..580)
                     /label=allpeg-R
     primer_bind     566..585
                     /label=allGFP-F
     promoter        597..1107
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1026..1044
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1116..1835
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1161..1182)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1422..1441)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1769..1790
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1958..2079
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1995..2014)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2049..2068
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2220..2237)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2391..2979)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2471..2490)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3150..4010)
                     /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     3773..3792
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4011..4115)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4142..4597
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4229..4248)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4439..4460
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4728..4776
                     /note="synthetic polyadenylation signal"
     misc_feature    4790..4881
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4830..4849
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     complement(4902..4921)
                     /label=allGFP-R
     primer_bind     4906..4925
                     /label=allpeg-F
     primer_bind     complement(4909..4928)
                     /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 ccttactcaa tctctgttcc gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcgaaa aggtgctccc
      361 agtaacagag attgagtaat ttttttaaag aattctcgac ctcgagacaa atggcagtat
      421 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag
      481 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc
      541 aaaattttcg ggtttattac agggacagca gagatccact ttggccgcgg ctcgaggggg
      601 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
      661 cgtggttccg ggaaacgcag cggcgccgac cctgggactc gcacattctt cacgtccgtt
      721 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
      781 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
      841 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
      901 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
      961 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
     1021 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
     1081 gaccgaatca ccgacctctc tccccagggg gatccatggt gagcaagggc gaggagctgt
     1141 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1201 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     1261 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1321 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1381 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     1441 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1501 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     1561 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     1621 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1681 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     1741 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     1801 ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgcgactct agatcataat
     1861 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     1921 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     1981 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2041 ttctagttgt ggtttgtcca aactcatcaa tgtatcttag tcgaccgatg cccttgagag
     2101 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     2161 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc
     2221 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2281 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2341 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2401 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2461 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2521 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2581 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2641 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2701 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2761 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2821 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2881 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2941 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3001 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3061 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3121 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3181 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3241 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg ggacccacgc
     3301 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3361 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3421 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3481 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3541 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3601 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3661 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3721 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3781 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3841 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3901 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3961 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4021 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4081 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4141 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4201 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4261 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4321 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4381 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4441 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4501 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4561 aacgcgaatt ttaacaaaat attaacgctt acaatttgcc attcgccatt caggctgcgc
     4621 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagcc caagctacca
     4681 tgataagtaa gtaatattaa ggtacgggag gtacttggag cggccgcaat aaaatatctt
     4741 tattttcatt acatctgtgt gttggttttt tgtgtgaatc gatagtacta acatacgctc
     4801 tccatcaaaa caaaacgaaa caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg
     4861 caggtgccag aacatttctc tatcgatagg taccgattag tgaacggatc tcgacggtat
     4921 cgatcacgag actagcctcg agcggccgcc cccttcacc
//
