LOCUS       pCas-Guide-EF1a-       10493 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Mammalian vector for co-expressing a guide RNA (gRNA) together with 
            Cas9 and TurboGFP, for genome editing using the S. pyogenes 
            CRISPR/Cas9 system.
ACCESSION   .
VERSION     .
KEYWORDS    pCas-Guide-EF1a-GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10493)
  AUTHORS   OriGene
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 10493)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        90..330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        384..459
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      460..465
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     promoter        480..1658
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     CDS             1737..2432
                     /label=TurboGFP
                     /note="improved green fluorescent protein from Pontellina 
                     plumata (Evdokimov et al., 2006)"
     polyA_signal    2464..2917
                     /label=PGK poly(A) signal
                     /note="mouse phosphoglycerate kinase 1 polyadenylation
                     signal"
     protein_bind    2944..2977
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        3047..3350
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3351..3554
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        3580..3598
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3657..7760
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             7761..7781
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             7788..7808
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             7830..7859
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
     CDS             7878..7901
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     polyA_signal    7948..8570
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     rep_origin      complement(8691..9279)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(9457..10314)
                     /label=AmpR
                     /note="beta-lactamase"
     primer_bind     10429..10445
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        10450..10468
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gaattcccca gtggaaagac gcgcaggcaa aacgcaccac gtgacggagc gtgaccgcgc
       61 gccgagcgcg cgccaaggtc gggcaggaag agggcctatt tcccatgatt ccttcatatt
      121 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa
      181 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
      241 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
      301 tcttgggttt atatatcttg tggaaaggac gcgggatcca ctggaccagg cagcagcgtc
      361 agaagacttt tttggaacgt ctcgttttag agctagaaat agcaagttaa aataaggcta
      421 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttggtg tacacgtgag
      481 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
      541 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
      601 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
      661 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
      721 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
      781 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
      841 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
      901 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
      961 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     1021 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     1081 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     1141 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     1201 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     1261 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     1321 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     1381 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     1441 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     1501 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     1561 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     1621 tggttcaaag tttttttctt ccatttcagg tgtcgtgact atagggcggc cggacgtgac
     1681 aaatggaagt agcacgcctc actaggctcg tgcagatgga cagcaccgct gcagccatgg
     1741 agagcgacga gagcggcctg cccgccatgg agatcgagtg ccgcatcacc ggcaccctga
     1801 acggcgtgga gttcgagctg gtgggcggcg gagagggcac ccccgagcag ggccgcatga
     1861 ccaacaagat gaagagcacc aaaggcgccc tgaccttcag cccctacctg ctgagccacg
     1921 tgatgggcta cggcttctac cacttcggca cctaccccag cggctacgag aaccccttcc
     1981 tgcacgccat caacaacggc ggctacacca acacccgcat cgagaagtac gaggacggcg
     2041 gcgtgctgca cgtgagcttc agctaccgct acgaggccgg ccgcgtgatc ggcgacttca
     2101 aggtgatggg caccggcttc cccgaggaca gcgtgatctt caccgacaag atcatccgca
     2161 gcaacgccac cgtggagcac ctgcacccca tgggcgataa cgatctggat ggcagcttca
     2221 cccgcacctt cagcctgcgc gacggcggct actacagctc cgtggtggac agccacatgc
     2281 acttcaagag cgccatccac cccagcatcc tgcagaacgg gggccccatg ttcgccttcc
     2341 gccgcgtgga ggaggatcac agcaacaccg agctgggcat cgtggagtac cagcacgcct
     2401 tcaagacccc ggatgcagat gccggtgaag aaagagttta atcgatgata tcagatcccc
     2461 gggatgcaga aattgatgat ctattaaaca ataaagatgt ccactaaaat ggaagttttt
     2521 cctgtcatac tttgttaaga agggtgagaa cagagtacct acattttgaa tggaaggatt
     2581 ggagctacgg gggtgggggt ggggtgggat tagataaatg cctgctcttt actgaaggct
     2641 ctttactatt gctttatgat aatgtttcat agttggatat cataatttaa acaagcaaaa
     2701 ccaaattaag ggccagctca ttcctcccac tcatgatcta tagatctata gatctctcgt
     2761 gggatcattg tttttctctt gattcccact ttgtggttct aagtactgtg gtttccaaat
     2821 gtgtcagttt catagcctga agaacgagat cagcagcctc tgttccacat acacttcatt
     2881 ctcagtattg ttttgccaag ttctaattcc atcagaagct ggtcgagatc cggaaccctt
     2941 aatataactt cgtataatgt atgctatacg aagttattag gtccactagt tattaatagt
     3001 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     3061 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     3121 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     3181 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta
     3241 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg
     3301 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     3361 tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     3421 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     3481 gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     3541 atataagcag agctcgttta gtgaaccgtc agaattttgt aatacgactc actatagggc
     3601 ggccgggaat tcgtcgactg gaaccggtac cgaggagatc tgccgccgcg atcgccatgg
     3661 ataagaaata ctcaatagga ctggatattg gcacaaatag cgtcggatgg gctgtgatca
     3721 ctgatgaata taaggttcct tctaaaaagt tcaaggttct gggaaataca gaccgccaca
     3781 gtatcaaaaa aaatcttata ggggctcttc tgtttgacag tggagagaca gccgaagcta
     3841 ctagactcaa acggacagct aggagaaggt atacaagacg gaagaatagg atttgttatc
     3901 tccaggagat tttttcaaat gagatggcca aagtggatga tagtttcttt catagacttg
     3961 aagagtcttt tttggtggaa gaagacaaga agcatgaaag acatcctatt tttggaaata
     4021 tagtggatga agttgcttat cacgagaaat atccaactat ctatcatctg agaaaaaaat
     4081 tggtggattc tactgataaa gccgatttgc gcctgatcta tttggccctg gcccacatga
     4141 ttaagtttag aggtcatttt ttgattgagg gcgatctgaa tcctgataat agtgatgtgg
     4201 acaaactgtt tatccagttg gtgcaaacct acaatcaact gtttgaagaa aaccctatta
     4261 acgcaagtgg agtggatgct aaagccattc tttctgcaag attgagtaaa tcaagaagac
     4321 tggaaaatct cattgctcag ctccccggtg agaagaaaaa tggcctgttt gggaatctca
     4381 ttgctttgtc attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg
     4441 ctaaactcca gctttcaaaa gatacttacg atgatgatct ggataatctg ttggctcaaa
     4501 ttggagatca atatgctgat ttgtttttgg cagctaagaa tctgtcagat gctattctgc
     4561 tttcagacat cctgagagtg aatactgaaa taactaaggc tcccctgtca gcttcaatga
     4621 ttaaacgcta cgatgaacat catcaagact tgactcttct gaaagccctg gttagacaac
     4681 aacttccaga aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt
     4741 atattgatgg cggcgcaagc caagaagaat tttataaatt tatcaaacca attctggaaa
     4801 aaatggatgg tactgaggaa ctgttggtga aactgaatag agaagatttg ctgcgcaagc
     4861 aacggacctt tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta
     4921 ttttgagaag acaagaagac ttttatccat ttctgaaaga caatagagag aagattgaaa
     4981 aaatcttgac ttttaggatt ccttattatg ttggtccatt ggccagaggc aatagtaggt
     5041 ttgcatggat gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg
     5101 tcgataaagg tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc
     5161 ttccaaatga aaaagtgctg ccaaaacata gtttgcttta tgagtatttt accgtttata
     5221 acgaattgac aaaggtcaaa tatgttactg aaggaatgag aaaaccagca tttctttcag
     5281 gtgaacagaa gaaagccatt gttgatctgc tcttcaaaac aaataggaaa gtgaccgtta
     5341 agcaactgaa agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag
     5401 gagttgaaga tagatttaat gcttcactgg gtacatacca tgatttgctg aaaattatta
     5461 aagataaaga ttttttggat aatgaagaaa atgaagacat cctggaggat attgttctga
     5521 cattgaccct gtttgaagat agggagatga ttgaggaaag acttaaaaca tacgctcacc
     5581 tctttgatga taaggtgatg aaacagctta aaagacgcag atatactggt tggggaaggt
     5641 tgtccagaaa attgattaat ggtattaggg ataagcaatc tggcaaaaca atactggatt
     5701 ttttgaaatc agatggtttt gccaatcgca attttatgca gctcatccat gatgatagtt
     5761 tgacatttaa agaagacatc caaaaagcac aagtgtctgg acaaggcgat agtctgcatg
     5821 aacatattgc aaatctggct ggtagccctg ctattaaaaa aggtattctc cagactgtga
     5881 aagttgttga tgaattggtc aaagtgatgg ggcggcataa gccagaaaat atcgttattg
     5941 aaatggcaag agaaaatcag acaactcaaa agggccagaa aaattccaga gagaggatga
     6001 aaagaatcga agaaggtatc aaagaactgg gaagtcagat tcttaaagag catcctgttg
     6061 aaaatactca attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagata
     6121 tgtatgtgga ccaagaactg gatattaata ggctgagtga ttatgatgtc gatcacattg
     6181 ttccacaaag tttccttaaa gacgattcaa tagacaataa ggtcctgacc aggtctgata
     6241 aaaatagagg taaatccgat aacgttccaa gtgaagaagt ggtcaaaaag atgaaaaact
     6301 attggagaca acttctgaac gccaagctga tcactcaaag gaagtttgat aatctgacca
     6361 aagctgaaag aggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg
     6421 ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta
     6481 aatacgatga aaatgataaa cttattagag aggttaaagt gattaccctg aaatctaaac
     6541 tggtttctga cttcagaaaa gatttccaat tctataaagt gagagagatt aacaattacc
     6601 atcatgccca tgatgcctat ctgaatgccg tcgttggaac tgctttgatt aagaaatatc
     6661 caaaacttga aagcgagttt gtctatggtg attataaagt ttatgatgtt aggaaaatga
     6721 ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa gtatttcttt tactctaata
     6781 tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc
     6841 tgatcgaaac taatggggaa actggagaaa ttgtctggga taaagggaga gattttgcca
     6901 cagtgcgcaa agtgttgtcc atgccccaag tcaatatcgt caagaaaaca gaagtgcaga
     6961 caggcggatt ctctaaggag tcaattctgc caaaaagaaa ttccgacaag ctgattgcta
     7021 ggaaaaaaga ctgggaccca aaaaaatatg gtggttttga tagtccaacc gtggcttatt
     7081 cagtcctggt ggttgctaag gtggaaaaag ggaaatccaa gaagctgaaa tccgttaaag
     7141 agctgctggg gatcacaatt atggaaagaa gttcctttga aaaaaatccc attgactttc
     7201 tggaagctaa aggatataag gaagttaaaa aagacctgat cattaaactg cctaaatata
     7261 gtctttttga gctggaaaac ggtaggaaac ggatgctggc tagtgccgga gaactgcaaa
     7321 aaggaaatga gctggctctg ccaagcaaat atgtgaattt tctgtatctg gctagtcatt
     7381 atgaaaagtt gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc
     7441 ataagcatta tctggatgag attattgagc aaatcagtga attttctaag agagttattc
     7501 tggcagatgc caatctggat aaagttctta gtgcatataa caaacataga gacaaaccaa
     7561 taagagaaca agcagaaaat atcattcatc tgtttacctt gaccaatctt ggagcacccg
     7621 ctgcttttaa atactttgat acaacaattg ataggaaaag atatacctct acaaaagaag
     7681 ttctggatgc cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt
     7741 tgagtcagct gggaggtgac cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc
     7801 ggaaagtgga cacgcgtacg cggccgctcg agcagaaact catctcagaa gaggatctgg
     7861 cagcaaatga tatcctggat tacaaggatg acgacgataa ggtttaaacg gccggccgcg
     7921 gtcatagctg tttcctgaac agatcccggg tggcatccct gtgacccctc cccagtgcct
     7981 ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt
     8041 tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta
     8101 tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca
     8161 agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg
     8221 attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc
     8281 taatttttgt ttttttggta gaggcggggt ttcaccatat tggccaggct ggtctccaac
     8341 tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga
     8401 accactgctc ccttccctgt ccttctgatt ttaaaataac tataccagca ggaggacgtc
     8461 cagacacagc ataggctacc tggccatgcc caaccggtgg gacatttgag ttgcttgctt
     8521 ggcactgtcc tctcatgcgt tgggtccact cagtagatgc ctgttgaatt gggtacgcgg
     8581 ccagcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     8641 ggataacgca ggaaagaaca tgtccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     8701 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     8761 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     8821 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     8881 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     8941 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     9001 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     9061 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     9121 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     9181 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     9241 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     9301 acggggtctg acgctcagtg gaacgacgcg taactcacgt taagggattt tggtcatgag
     9361 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     9421 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     9481 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     9541 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     9601 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     9661 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     9721 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     9781 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     9841 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     9901 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     9961 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
    10021 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
    10081 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
    10141 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
    10201 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
    10261 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
    10321 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgatga tattatttta
    10381 tcttgtgcaa tgtaacatca gagattttga gacacgggcc agagctgcca ggaaacagct
    10441 atgaccatgt aatacgactc actatagggg atatcagctg gatggcagtt aac
//
