LOCUS       pGL3-Ctr2-gRNA-EGFP     4928 bp ds-DNA     circular     19-AUG-2024
DEFINITION  .
KEYWORDS    "accession:addgene_107721_208561"
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
COMMENT     Imported from database: AddGene Entry: pGL3-U6-sgRNA-EGFP URL:
            https://www.addgene.org/browse/sequence/208561/ GENERAL Depositing
            Lab: Xingxu Huang Publication: Liu et al Nat Commun. 2018 Jun
            14;9(1):2338. doi: 10.1038/s41467-018-04768-7. Purpose: (Empty
            Backbone) For expression of sgRNA from the U6 promoter with EGFP
            expression simultaneously BACKBONE Vector backbone:
            pGL3-U6-sgRNA-PGK-puromycin Vector type: CRISPR GROWTH IN BACTERIA
            Bacterial Resistance(s): Ampicillin Copy number: High Copy Growth
            Strain(s): DH5alpha Growth Temperature: 37°C GENE/INSERT
            Gene/Insert name: None
FEATURES             Location/Qualifiers
     primer_bind     1..21
                     /label="hU6-F"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
     promoter        1..241
                     /label="U6 promoter"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
     primer_bind     172..191
                     /label="LKO.1 5'"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
     misc_feature    251..272
                     /label="Ctr2-gRNA"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     misc_RNA        273..348
                     /label="gRNA scaffold"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     misc_feature    400..517
                     /label="cPPT/CTS"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
     promoter        566..1076
                     /label="hPGK promoter"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
     primer_bind     995..1013
                     /label="hPGK-F"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
     CDS             1085..1804
                     /label="EGFP"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     primer_bind     complement(1130..1151)
                     /label="EGFP-N"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     primer          1220..1238
                     /label="eGFP F (Sarah)"
                     /note="sequence: aagttcatctgcaccaccg"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     primer          complement(1373..1392)
                     /label="eGFP R (Sarah)"
                     /note="sequence: tccttgaagaagatggtgcg"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     primer_bind     complement(1391..1410)
                     /label="EXFP-R"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     primer          complement(1625..1644)
                     /label="eGFP R2 (ZJ)"
                     /note="sequence: gtgttctgctggtagtggtc"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     primer_bind     1738..1759
                     /label="EGFP-C"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
     polyA_signal    1927..2048
                     /label="SV40 poly(A) signal"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
     primer_bind     complement(1964..1983)
                     /label="SV40pA-R"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     primer_bind     2018..2037
                     /label="EBV-rev"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
     primer_bind     complement(2189..2206)
                     /label="L4440"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
     rep_origin      complement(2360..2948)
                     /label="ori"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
     primer_bind     complement(2440..2459)
                     /label="pBR322ori-F"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
     CDS             complement(3119..3979)
                     /label="AmpR"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     primer_bind     3742..3761
                     /label="Amp-R"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     promoter        complement(3980..4084)
                     /label="AmpR promoter"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     rep_origin      4111..4566
                     /label="f1 ori"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     primer_bind     complement(4198..4217)
                     /label="F1ori-R"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     primer_bind     4408..4429
                     /label="F1ori-F"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     polyA_signal    4697..4745
                     /label="synthetic polyadenylation signal"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     misc_feature    4759..4850
                     /label="pause site"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     primer_bind     4799..4818
                     /label="RVprimer3"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
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 GTGCGATGGG GGGGTGGGTA GCgttttaga gctagaaata gcaagttaaa
      301 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttaaaga
      361 attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
      421 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
      481 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
      541 agatccactt tggccgcggc tcgagggggt tggggttgcg ccttttccaa ggcagccctg
      601 ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc
      661 ctgggactcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc
      721 ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt
      781 tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc ctcgcagacg
      841 gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct
      901 gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg
      961 gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg
     1021 agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct ccccaggggg
     1081 atccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1141 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     1201 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1261 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1321 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1381 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1441 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1501 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1561 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1621 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1681 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1741 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1801 gtaaagcggc cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt
     1861 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     1921 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     1981 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2041 gtatcttagt cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc
     2101 gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg
     2161 acaggtgccg gcagcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2221 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2281 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2341 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2401 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2461 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2521 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2581 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2641 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2701 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2761 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2821 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2881 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2941 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3001 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3061 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3121 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3181 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3241 gtgctgcaat gataccgcgg gacccacgct caccggctcc agatttatca gcaataaacc
     3301 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3361 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3421 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3481 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3541 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3601 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3661 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3721 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3781 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3841 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3901 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     3961 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4021 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4081 cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg
     4141 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4201 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4261 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4321 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4381 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4441 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4501 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     4561 caatttgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     4621 cttcgctatt acgccagccc aagctaccat gataagtaag taatattaag gtacgggagg
     4681 tacttggagc ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt
     4741 gtgtgaatcg atagtactaa catacgctct ccatcaaaac aaaacgaaac aaaacaaact
     4801 agcaaaatag gctgtcccca gtgcaagtgc aggtgccaga acatttctct atcgataggt
     4861 accgattagt gaacggatct cgacggtatc gatcacgaga ctagcctcga gcggccgccc
     4921 ccttcacc
//