LOCUS       pCas-Guide-EF1a-       11006 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Mammalian vector for co-expressing a guide RNA (gRNA) together with 
            Cas9 and CD4, for genome editing using the S. pyogenes CRISPR/Cas9 
            system.
ACCESSION   .
VERSION     .
KEYWORDS    pCas-Guide-EF1a-CD4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11006)
  AUTHORS   OriGene
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 11006)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..11006
                     /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             1679..2950
                     /label=CD4 (truncated)
                     /note="truncated human CD4 cell surface glycoprotein
                     lacking the cytoplasmic tail"
     polyA_signal    2977..3430
                     /label=PGK poly(A) signal
                     /note="mouse phosphoglycerate kinase 1 polyadenylation
                     signal"
     protein_bind    3457..3490
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        3560..3863
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3864..4067
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        4093..4111
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4170..8273
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             8274..8294
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             8301..8321
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             8343..8372
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
     CDS             8391..8414
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     polyA_signal    8461..9083
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     rep_origin      complement(9204..9792)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(9970..10827)
                     /label=AmpR
                     /note="beta-lactamase"
     primer_bind     10942..10958
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        10963..10981
                     /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 cggacgtgat
     1681 gaaccgggga gtccctttta ggcacttgct tctggtgctg caactggcgc tcctcccagc
     1741 agccactcag ggaaagaaag tggtgctggg caaaaaaggg gatacagtgg aactgacctg
     1801 tacagcttcc cagaagaaga gcatacaatt ccactggaaa aactccaacc agataaagat
     1861 tctgggaaat cagggctcct tcttaactaa aggtccatcc aagctgaatg atcgcgctga
     1921 ctcaagaaga agcctttggg accaaggaaa ctttcccctg atcatcaaga atcttaagat
     1981 agaagactca gatacttaca tctgtgaagt ggaggaccag aaggaggagg tgcaattgct
     2041 agtgttcgga ttgactgcca actctgacac ccacctgctt caggggcaga gcctgaccct
     2101 gaccttggag agcccccctg gtagtagccc ctcagtgcaa tgtaggagtc caaggggtaa
     2161 aaacatacag ggggggaaga ccctctccgt gtctcagctg gagctccagg atagtggcac
     2221 ctggacatgc actgtcttgc agaaccagaa gaaggtggag ttcaaaatag acatcgtggt
     2281 gctagctttc cagaaggcct ccagcatagt ctataagaaa gagggggaac aggtggagtt
     2341 ctccttccca ctcgccttta cagttgaaaa gctgacgggc agtggcgagc tgtggtggca
     2401 ggcggagagg gcttcctcct ccaagtcttg gatcaccttt gacctgaaga acaaggaagt
     2461 gtctgtaaaa cgggttaccc aggaccctaa gctccagatg ggcaagaagc tcccgctcca
     2521 cctcaccctg ccccaggcct tgcctcagta tgctggctct ggaaacctca ccctggccct
     2581 tgaagcgaaa acaggaaagt tgcatcagga agtgaacctg gtggtgatga gagccactca
     2641 gctccagaaa aatttgacct gtgaggtgtg gggacccacc tcccctaagc tgatgctgag
     2701 cttgaaactg gagaacaagg aggcaaaggt ctcgaagcgg gagaaggcgg tgtgggtgct
     2761 gaaccctgag gcggggatgt ggcagtgtct gctgagtgac tcgggacagg tcctgctgga
     2821 atccaacatc aaggttctgc ccacatggtc caccccggtg cagccaatgg ccctgattgt
     2881 gctggggggc gtcgccggcc tcctgctttt cattgggcta ggcatcttct tctgtgtcag
     2941 gtgccggcac accggttagg atatcagatc cccgggatgc agaaattgat gatctattaa
     3001 acaataaaga tgtccactaa aatggaagtt tttcctgtca tactttgtta agaagggtga
     3061 gaacagagta cctacatttt gaatggaagg attggagcta cgggggtggg ggtggggtgg
     3121 gattagataa atgcctgctc tttactgaag gctctttact attgctttat gataatgttt
     3181 catagttgga tatcataatt taaacaagca aaaccaaatt aagggccagc tcattcctcc
     3241 cactcatgat ctatagatct atagatctct cgtgggatca ttgtttttct cttgattccc
     3301 actttgtggt tctaagtact gtggtttcca aatgtgtcag tttcatagcc tgaagaacga
     3361 gatcagcagc ctctgttcca catacacttc attctcagta ttgttttgcc aagttctaat
     3421 tccatcagaa gctggtcgag atccggaacc cttaatataa cttcgtataa tgtatgctat
     3481 acgaagttat taggtccact agttattaat agtaatcaat tacggggtca ttagttcata
     3541 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     3601 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     3661 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     3721 atcaagtgta tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg
     3781 cctggcatta tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg
     3841 tattagtcat cgctattacc atggtgatgc ggttttggca gtacaccaat gggcgtggat
     3901 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     3961 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc
     4021 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc
     4081 gtcagaattt tgtaatacga ctcactatag ggcggccggg aattcgtcga ctggaaccgg
     4141 taccgaggag atctgccgcc gcgatcgcca tggataagaa atactcaata ggactggata
     4201 ttggcacaaa tagcgtcgga tgggctgtga tcactgatga atataaggtt ccttctaaaa
     4261 agttcaaggt tctgggaaat acagaccgcc acagtatcaa aaaaaatctt ataggggctc
     4321 ttctgtttga cagtggagag acagccgaag ctactagact caaacggaca gctaggagaa
     4381 ggtatacaag acggaagaat aggatttgtt atctccagga gattttttca aatgagatgg
     4441 ccaaagtgga tgatagtttc tttcatagac ttgaagagtc ttttttggtg gaagaagaca
     4501 agaagcatga aagacatcct atttttggaa atatagtgga tgaagttgct tatcacgaga
     4561 aatatccaac tatctatcat ctgagaaaaa aattggtgga ttctactgat aaagccgatt
     4621 tgcgcctgat ctatttggcc ctggcccaca tgattaagtt tagaggtcat tttttgattg
     4681 agggcgatct gaatcctgat aatagtgatg tggacaaact gtttatccag ttggtgcaaa
     4741 cctacaatca actgtttgaa gaaaacccta ttaacgcaag tggagtggat gctaaagcca
     4801 ttctttctgc aagattgagt aaatcaagaa gactggaaaa tctcattgct cagctccccg
     4861 gtgagaagaa aaatggcctg tttgggaatc tcattgcttt gtcattgggt ttgaccccta
     4921 attttaaatc aaattttgat ttggcagaag atgctaaact ccagctttca aaagatactt
     4981 acgatgatga tctggataat ctgttggctc aaattggaga tcaatatgct gatttgtttt
     5041 tggcagctaa gaatctgtca gatgctattc tgctttcaga catcctgaga gtgaatactg
     5101 aaataactaa ggctcccctg tcagcttcaa tgattaaacg ctacgatgaa catcatcaag
     5161 acttgactct tctgaaagcc ctggttagac aacaacttcc agaaaagtat aaagaaatct
     5221 tttttgatca atcaaaaaac ggatatgcag gttatattga tggcggcgca agccaagaag
     5281 aattttataa atttatcaaa ccaattctgg aaaaaatgga tggtactgag gaactgttgg
     5341 tgaaactgaa tagagaagat ttgctgcgca agcaacggac ctttgacaac ggctctattc
     5401 cccatcaaat tcacttgggt gagctgcatg ctattttgag aagacaagaa gacttttatc
     5461 catttctgaa agacaataga gagaagattg aaaaaatctt gacttttagg attccttatt
     5521 atgttggtcc attggccaga ggcaatagta ggtttgcatg gatgactcgg aagtctgaag
     5581 aaacaattac cccatggaat tttgaagaag ttgtcgataa aggtgcttca gctcaatcat
     5641 ttattgaacg catgacaaac tttgataaaa atcttccaaa tgaaaaagtg ctgccaaaac
     5701 atagtttgct ttatgagtat tttaccgttt ataacgaatt gacaaaggtc aaatatgtta
     5761 ctgaaggaat gagaaaacca gcatttcttt caggtgaaca gaagaaagcc attgttgatc
     5821 tgctcttcaa aacaaatagg aaagtgaccg ttaagcaact gaaagaagat tatttcaaaa
     5881 aaatagaatg ttttgatagt gttgaaattt caggagttga agatagattt aatgcttcac
     5941 tgggtacata ccatgatttg ctgaaaatta ttaaagataa agattttttg gataatgaag
     6001 aaaatgaaga catcctggag gatattgttc tgacattgac cctgtttgaa gatagggaga
     6061 tgattgagga aagacttaaa acatacgctc acctctttga tgataaggtg atgaaacagc
     6121 ttaaaagacg cagatatact ggttggggaa ggttgtccag aaaattgatt aatggtatta
     6181 gggataagca atctggcaaa acaatactgg attttttgaa atcagatggt tttgccaatc
     6241 gcaattttat gcagctcatc catgatgata gtttgacatt taaagaagac atccaaaaag
     6301 cacaagtgtc tggacaaggc gatagtctgc atgaacatat tgcaaatctg gctggtagcc
     6361 ctgctattaa aaaaggtatt ctccagactg tgaaagttgt tgatgaattg gtcaaagtga
     6421 tggggcggca taagccagaa aatatcgtta ttgaaatggc aagagaaaat cagacaactc
     6481 aaaagggcca gaaaaattcc agagagagga tgaaaagaat cgaagaaggt atcaaagaac
     6541 tgggaagtca gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc
     6601 tctatctcta ttatctccaa aatggaagag atatgtatgt ggaccaagaa ctggatatta
     6661 ataggctgag tgattatgat gtcgatcaca ttgttccaca aagtttcctt aaagacgatt
     6721 caatagacaa taaggtcctg accaggtctg ataaaaatag aggtaaatcc gataacgttc
     6781 caagtgaaga agtggtcaaa aagatgaaaa actattggag acaacttctg aacgccaagc
     6841 tgatcactca aaggaagttt gataatctga ccaaagctga aagaggaggt ttgagtgaac
     6901 ttgataaagc tggttttatc aaacgccaat tggttgaaac tcgccaaatc actaagcatg
     6961 tggcacaaat tttggatagt cgcatgaata ctaaatacga tgaaaatgat aaacttatta
     7021 gagaggttaa agtgattacc ctgaaatcta aactggtttc tgacttcaga aaagatttcc
     7081 aattctataa agtgagagag attaacaatt accatcatgc ccatgatgcc tatctgaatg
     7141 ccgtcgttgg aactgctttg attaagaaat atccaaaact tgaaagcgag tttgtctatg
     7201 gtgattataa agtttatgat gttaggaaaa tgattgctaa gtctgagcaa gaaataggca
     7261 aagcaaccgc aaagtatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta
     7321 cacttgcaaa tggagagatt cgcaaacgcc ctctgatcga aactaatggg gaaactggag
     7381 aaattgtctg ggataaaggg agagattttg ccacagtgcg caaagtgttg tccatgcccc
     7441 aagtcaatat cgtcaagaaa acagaagtgc agacaggcgg attctctaag gagtcaattc
     7501 tgccaaaaag aaattccgac aagctgattg ctaggaaaaa agactgggac ccaaaaaaat
     7561 atggtggttt tgatagtcca accgtggctt attcagtcct ggtggttgct aaggtggaaa
     7621 aagggaaatc caagaagctg aaatccgtta aagagctgct ggggatcaca attatggaaa
     7681 gaagttcctt tgaaaaaaat cccattgact ttctggaagc taaaggatat aaggaagtta
     7741 aaaaagacct gatcattaaa ctgcctaaat atagtctttt tgagctggaa aacggtagga
     7801 aacggatgct ggctagtgcc ggagaactgc aaaaaggaaa tgagctggct ctgccaagca
     7861 aatatgtgaa ttttctgtat ctggctagtc attatgaaaa gttgaagggt agtccagaag
     7921 ataacgaaca aaaacaattg tttgtggagc agcataagca ttatctggat gagattattg
     7981 agcaaatcag tgaattttct aagagagtta ttctggcaga tgccaatctg gataaagttc
     8041 ttagtgcata taacaaacat agagacaaac caataagaga acaagcagaa aatatcattc
     8101 atctgtttac cttgaccaat cttggagcac ccgctgcttt taaatacttt gatacaacaa
     8161 ttgataggaa aagatatacc tctacaaaag aagttctgga tgccactctt atccatcaat
     8221 ccatcactgg tctttatgaa acacgcattg atttgagtca gctgggaggt gaccccaaga
     8281 aaaaacgcaa ggtggaagat cctaagaaaa agcggaaagt ggacacgcgt acgcggccgc
     8341 tcgagcagaa actcatctca gaagaggatc tggcagcaaa tgatatcctg gattacaagg
     8401 atgacgacga taaggtttaa acggccggcc gcggtcatag ctgtttcctg aacagatccc
     8461 gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca
     8521 gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
     8581 ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca
     8641 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg
     8701 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg
     8761 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagaggcgg
     8821 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct
     8881 tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg
     8941 attttaaaat aactatacca gcaggaggac gtccagacac agcataggct acctggccat
     9001 gcccaaccgg tgggacattt gagttgcttg cttggcactg tcctctcatg cgttgggtcc
     9061 actcagtaga tgcctgttga attgggtacg cggccagcgg cgagcggtat cagctcactc
     9121 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtccgt
     9181 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     9241 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     9301 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     9361 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     9421 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     9481 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     9541 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     9601 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     9661 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     9721 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     9781 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgac
     9841 gcgtaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     9901 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     9961 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
    10021 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
    10081 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
    10141 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
    10201 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
    10261 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
    10321 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
    10381 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
    10441 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
    10501 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
    10561 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
    10621 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
    10681 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
    10741 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
    10801 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
    10861 tcagggttat tgtctcatga tgatattatt ttatcttgtg caatgtaaca tcagagattt
    10921 tgagacacgg gccagagctg ccaggaaaca gctatgacca tgtaatacga ctcactatag
    10981 gggatatcag ctggatggca gttaac
//
