LOCUS       Exported                4954 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 4954)
  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 4954)
  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..4954
                     /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    426..543
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     554..573
                     /label=newallGFP-F
     primer_bind     complement(556..575)
                     /label=allpeg-R
     primer_bind     561..580
                     /label=allGFP-F
     promoter        592..1102
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1021..1039
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1111..1830
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1156..1177)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1417..1436)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1764..1785
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1953..2074
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1990..2009)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2044..2063
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2215..2232)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2386..2974)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2466..2485)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3145..4005)
                     /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     3768..3787
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4006..4110)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4137..4592
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4224..4243)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4434..4455
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4723..4771
                     /note="synthetic polyadenylation signal"
     misc_feature    4785..4876
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4825..4844
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     complement(4897..4916)
                     /label=allGFP-R
     primer_bind     4901..4920
                     /label=allpeg-F
     primer_bind     complement(4904..4923)
                     /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 gaggcataca tctccatgga gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcgact ggtagacctc
      361 catggagatg tatgtttttt taaagaattc tcgacctcga gacaaatggc agtattcatc
      421 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
      481 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
      541 tttcgggttt attacaggga cagcagagat ccactttggc cgcggctcga gggggttggg
      601 gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg
      661 ttccgggaaa cgcagcggcg ccgaccctgg gactcgcaca ttcttcacgt ccgttcgcag
      721 cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc
      781 cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc
      841 acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc
      901 gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga
      961 aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg
     1021 gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg
     1081 aatcaccgac ctctctcccc agggggatcc atggtgagca agggcgagga gctgttcacc
     1141 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1201 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1261 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1321 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1381 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1441 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1501 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1561 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1621 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1681 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     1741 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1801 actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc ataatcagcc
     1861 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     1921 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     1981 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2041 gttgtggttt gtccaaactc atcaatgtat cttagtcgac cgatgccctt gagagccttc
     2101 aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact
     2161 gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctcttccg cttcctcgct
     2221 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2281 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2341 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2401 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2461 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2521 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2581 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2641 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2701 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     2761 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     2821 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     2881 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     2941 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3001 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3061 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3121 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3181 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3241 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgggacc cacgctcacc
     3301 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3361 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3421 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     3481 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     3541 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     3601 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3661 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3721 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     3781 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     3841 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     3901 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     3961 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4021 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4081 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc
     4141 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4201 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4261 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4321 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     4381 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4441 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4501 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4561 gaattttaac aaaatattaa cgcttacaat ttgccattcg ccattcaggc tgcgcaactg
     4621 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagcccaagc taccatgata
     4681 agtaagtaat attaaggtac gggaggtact tggagcggcc gcaataaaat atctttattt
     4741 tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag tactaacata cgctctccat
     4801 caaaacaaaa cgaaacaaaa caaactagca aaataggctg tccccagtgc aagtgcaggt
     4861 gccagaacat ttctctatcg ataggtaccg attagtgaac ggatctcgac ggtatcgatc
     4921 acgagactag cctcgagcgg ccgccccctt cacc
//
