LOCUS       P-pU6_5'-CMV,_EF1-alpha 9678 bp ds-DNA     circular     15-SEP-2021
DEFINITION  .
KEYWORDS    "Parent Construct" "Plasmid Type:dual guide" "Gene Insert" "Cas"   
            "Domain" "Antibiotic Resistance:Ampicillin" "Mammalian Resistance"
            "Promoter:pU6 5'-CMV, EF1-alpha" "Fluorescence Cassette:mTagBFP2"
            "Guide Constant Region" "Notes"
FEATURES             Location/Qualifiers
     misc_feature    263..826
                     /label="pU6 5'-CMV"
     promoter        323..626
                     /label="CMV enhancer"
     misc_recomb     2825..2858
                     /label="LoxP"
     misc_feature    2945..2964
                     /label="eGFP-NT2"
     misc_feature    2965..3083
                     /label="Cas9 Constant Region1-CS1"
     misc_feature    3813..4023
                     /label="EF1-alpha"
     misc_feature    5745..6458
                     /label="mTagBFP2"
     misc_recomb     6478..6511
                     /label="LoxP"
     rep_origin      complement(7908..8590)
                     /label="ColE1 origin"
     CDS             complement(8688..9347)
                     /label="AmpR"
     misc_feature    complement(8688..9347)
                     /label="Ampicillin"
ORIGIN
        1 gcttaagcgg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt
      841 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      901 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      961 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     1021 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
     1081 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
     1141 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
     1201 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
     1261 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
     1321 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
     1381 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
     1441 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
     1501 agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgcgctgat cttcagacct
     1561 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     1621 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     1681 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     1741 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     1801 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     1861 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     1921 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     1981 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     2041 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     2101 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     2161 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     2221 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     2281 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc
     2341 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac
     2401 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa
     2461 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     2521 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     2581 ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctaga gatccgacgc
     2641 gccatctcta ggcccgcgcc ggccccctcg cacggacttg tgggagaagc tcggctactc
     2701 ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct taatgtgcga
     2761 taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat aggcttggat
     2821 ttctataact tcgtatagca tacattatac gaagttataa acagcacaaa aggaaactca
     2881 ccctaactgt aaagtaattg tgtgttttga gactataagt atcccttgga gaaccacctt
     2941 gttggaccag gatgggcacc acccgtttaa gagctaagct ggaaacagca tagcaagttt
     3001 aaataaggct agtccgttat caacttggcc gctttaaggc cggtcctagc aaggccaagt
     3061 ggcaccgagt cggtgctttt tttctcgagt actaggatcc attaggcggc cgccgggagg
     3121 tggtccctgc agttacgcca atgataaccc ccgccagaaa aatcttagta gccttccctt
     3181 tttgttttcc gtgccccaac tcggcggatt gactcggccc cttccggaaa cacccgaatc
     3241 aacttctagt caaattattg ttcacgccgc aatgacccac ccctggcccg cgtctgtgga
     3301 actgacccct ggtgtacagg agagttcgct gctgaaagtg gtcccaaagg ggtactagtt
     3361 tttaagctcc caactccccc tcccccagcg tctggaggat tccacaccct cgcaccgcag
     3421 gggcgaggaa gtgggcggag tccggttttg gcgccagccg ctgaggctgc caagcagaaa
     3481 agccaccgct gaggagactc cggtcactgt cctcgccccg cctccccctt ccctcccctt
     3541 ggggaccacc gggcgccacg ccgcgaacgg taagtgccgc ggtcgtcggc gcctccgccc
     3601 tccccctagg gccccaattc ccagcgggcg cggcgcgcgg cccctccccc cgccgggcgc
     3661 gcgcccgctg ccccgccctt cgtggccgcc cggcgtgggc ggtgccaccc ctccccccgg
     3721 cggcccccct gcagccgcgc gcagctcccg gctccctccc ccttcggatg tggcttgagc
     3781 tgtaggcgcg gagatgcatg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
     3841 gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta
     3901 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     3961 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     4021 caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc
     4081 gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt
     4141 ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga
     4201 gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc
     4261 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg
     4321 ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg
     4381 gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg
     4441 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct
     4501 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc
     4561 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc
     4621 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa
     4681 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc
     4741 aggcacctcg attagttctc gcgcttttgg agtacgtcgt ctttaggttg gggggagggg
     4801 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg
     4861 cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc
     4921 aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg agctagccca
     4981 ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg
     5041 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgacccgg
     5101 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     5161 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     5221 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     5281 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     5341 agcccgcgtg gttcctggcc accgtcggcg tgtcgcccga ccaccagggc aagggtctgg
     5401 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     5461 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     5521 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg
     5581 gatcgggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc
     5641 caccggtccc tgccgctaaa cgggtcaaac tcgacggcgg ttctggaggt cctgctgcaa
     5701 agcgcgttaa gttggatgga gggtcagggg gagccaccgc caccatggtg tctaagggcg
     5761 aagagctgat taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc
     5821 atcacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga
     5881 gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct
     5941 tcctctacgg cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc
     6001 agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc
     6061 tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca
     6121 gaggggtgaa cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg
     6181 ccttcaccga gacCctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc
     6241 tgaagctcgt gggcgggagc catctgatcg caaacgccaa gaccacatat agatccaaga
     6301 aacccgctaa gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa
     6361 tcaaggaggc caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact
     6421 gcgacctccc tagcaaactg gggcacaagc ttaattaaga attcgtcgag ggacctaata
     6481 acttcgtata gcatacatta tacgaagtta tacatgttta agggttccgg ttccactagg
     6541 tacaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     6601 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     6661 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     6721 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     6781 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     6841 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     6901 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     6961 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     7021 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     7081 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     7141 gggccgcctc cccgcatcga taccgtcgac ctcgatcgag acctagaaaa acatggagca
     7201 atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag
     7261 gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag
     7321 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     7381 tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct
     7441 gattggcaga actacacacc agggccaggg atcagatatc cactgacctt tggatggtgc
     7501 tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg agagaacacc
     7561 cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga agtattagag
     7621 tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca tccggactgt
     7681 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     7741 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     7801 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     7861 agcagcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     7921 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     7981 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     8041 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     8101 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     8161 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     8221 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     8281 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     8341 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     8401 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     8461 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     8521 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     8581 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     8641 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     8701 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     8761 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     8821 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     8881 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     8941 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     9001 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     9061 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     9121 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     9181 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     9241 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     9301 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     9361 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     9421 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     9481 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     9541 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     9601 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     9661 cgaaaagtgc cacctgac
//