pCas-Guide-CRISPRi

LOCUS       pCas-Guide-CRISPRi        9198 bp DNA     circular SYN 03-NOV-2024
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9198)
  AUTHORS   Ming
  TITLE     Direct Submission
  JOURNAL   Exported Nov 3, 2024 from SnapGene 6.0.2 to Vector NTI(R) format
            https://www.biofeng.com
COMMENT     VNTDATE|1073001600|
COMMENT     VNTDBDATE|1073001600|
COMMENT     VNTNAME|pCas-Guide-CRISPRi|
COMMENT     VNTAUTHORNAME|Ming|
FEATURES             Location/Qualifiers
     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"
     enhancer        511..890
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        891..1094
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        1120..1138
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1197..5300
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
     CDS             5301..5321
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
     CDS             5328..5348
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
     CDS             5409..5603
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
     CDS             5637..5657
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
     CDS             5667..6524
                     /codon_start=1
                     /product="transcriptional repression domain from rat 
                     methyl-CpG-binding protein 2 (Nan et al., 1997)"
                     /label=MeCP2
     CDS             6546..6575
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
     CDS             6594..6617
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
     primer_bind     complement(6637..6653)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    6664..7286
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     rep_origin      complement(7407..7995)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8159..9019)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
     primer_bind     9134..9150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        9155..9173
                     /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 tacatttata
      481 ttggctcatg tccaatatga ccgccatgtt gacattgatt attgactagt tattaatagt
      541 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      601 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      661 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      721 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta
      781 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg
      841 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      901 tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      961 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     1021 gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     1081 atataagcag agctcgttta gtgaaccgtc agaattttgt aatacgactc actatagggc
     1141 ggccgggaat tcgtcgactg gaaccggtac cgaggagatc tgccgccgcg atcgccatgg
     1201 ataagaaata ctcaatagga ctggctattg gcacaaatag cgtcggatgg gctgtgatca
     1261 ctgatgaata taaggttcct tctaaaaagt tcaaggttct gggaaataca gaccgccaca
     1321 gtatcaaaaa aaatcttata ggggctcttc tgtttgacag tggagagaca gccgaagcta
     1381 ctagactcaa acggacagct aggagaaggt atacaagacg gaagaatagg atttgttatc
     1441 tccaggagat tttttcaaat gagatggcca aagtggatga tagtttcttt catagacttg
     1501 aagagtcttt tttggtggaa gaagacaaga agcatgaaag acatcctatt tttggaaata
     1561 tagtggatga agttgcttat cacgagaaat atccaactat ctatcatctg agaaaaaaat
     1621 tggtggattc tactgataaa gccgatttgc gcctgatcta tttggccctg gcccacatga
     1681 ttaagtttag aggtcatttt ttgattgagg gcgatctgaa tcctgataat agtgatgtgg
     1741 acaaactgtt tatccagttg gtgcaaacct acaatcaact gtttgaagaa aaccctatta
     1801 acgcaagtgg agtggatgct aaagccattc tttctgcaag attgagtaaa tcaagaagac
     1861 tggaaaatct cattgctcag ctccccggtg agaagaaaaa tggcctgttt gggaatctca
     1921 ttgctttgtc attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg
     1981 ctaaactcca gctttcaaaa gatacttacg atgatgatct ggataatctg ttggctcaaa
     2041 ttggagatca atatgctgat ttgtttttgg cagctaagaa tctgtcagat gctattctgc
     2101 tttcagacat cctgagagtg aatactgaaa taactaaggc tcccctgtca gcttcaatga
     2161 ttaaacgcta cgatgaacat catcaagact tgactcttct gaaagccctg gttagacaac
     2221 aacttccaga aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt
     2281 atattgatgg cggcgcaagc caagaagaat tttataaatt tatcaaacca attctggaaa
     2341 aaatggatgg tactgaggaa ctgttggtga aactgaatag agaagatttg ctgcgcaagc
     2401 aacggacctt tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta
     2461 ttttgagaag acaagaagac ttttatccat ttctgaaaga caatagagag aagattgaaa
     2521 aaatcttgac ttttaggatt ccttattatg ttggtccatt ggccagaggc aatagtaggt
     2581 ttgcatggat gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg
     2641 tcgataaagg tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc
     2701 ttccaaatga aaaagtgctg ccaaaacata gtttgcttta tgagtatttt accgtttata
     2761 acgaattgac aaaggtcaaa tatgttactg aaggaatgag aaaaccagca tttctttcag
     2821 gtgaacagaa gaaagccatt gttgatctgc tcttcaaaac aaataggaaa gtgaccgtta
     2881 agcaactgaa agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag
     2941 gagttgaaga tagatttaat gcttcactgg gtacatacca tgatttgctg aaaattatta
     3001 aagataaaga ttttttggat aatgaagaaa atgaagacat cctggaggat attgttctga
     3061 cattgaccct gtttgaagat agggagatga ttgaggaaag acttaaaaca tacgctcacc
     3121 tctttgatga taaggtgatg aaacagctta aaagacgcag atatactggt tggggaaggt
     3181 tgtccagaaa attgattaat ggtattaggg ataagcaatc tggcaaaaca atactggatt
     3241 ttttgaaatc agatggtttt gccaatcgca attttatgca gctcatccat gatgatagtt
     3301 tgacatttaa agaagacatc caaaaagcac aagtgtctgg acaaggcgat agtctgcatg
     3361 aacatattgc aaatctggct ggtagccctg ctattaaaaa aggtattctc cagactgtga
     3421 aagttgttga tgaattggtc aaagtgatgg ggcggcataa gccagaaaat atcgttattg
     3481 aaatggcaag agaaaatcag acaactcaaa agggccagaa aaattccaga gagaggatga
     3541 aaagaatcga agaaggtatc aaagaactgg gaagtcagat tcttaaagag catcctgttg
     3601 aaaatactca attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagata
     3661 tgtatgtgga ccaagaactg gatattaata ggctgagtga ttatgatgtc gatgccattg
     3721 ttccacaaag tttccttaaa gacgattcaa tagacaataa ggtcctgacc aggtctgata
     3781 aaaatagagg taaatccgat aacgttccaa gtgaagaagt ggtcaaaaag atgaaaaact
     3841 attggagaca acttctgaac gccaagctga tcactcaaag gaagtttgat aatctgacca
     3901 aagctgaaag aggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg
     3961 ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta
     4021 aatacgatga aaatgataaa cttattagag aggttaaagt gattaccctg aaatctaaac
     4081 tggtttctga cttcagaaaa gatttccaat tctataaagt gagagagatt aacaattacc
     4141 atcatgccca tgatgcctat ctgaatgccg tcgttggaac tgctttgatt aagaaatatc
     4201 caaaacttga aagcgagttt gtctatggtg attataaagt ttatgatgtt aggaaaatga
     4261 ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa gtatttcttt tactctaata
     4321 tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc
     4381 tgatcgaaac taatggggaa actggagaaa ttgtctggga taaagggaga gattttgcca
     4441 cagtgcgcaa agtgttgtcc atgccccaag tcaatatcgt caagaaaaca gaagtgcaga
     4501 caggcggatt ctctaaggag tcaattctgc caaaaagaaa ttccgacaag ctgattgcta
     4561 ggaaaaaaga ctgggaccca aaaaaatatg gtggttttga tagtccaacc gtggcttatt
     4621 cagtcctggt ggttgctaag gtggaaaaag ggaaatccaa gaagctgaaa tccgttaaag
     4681 agctgctggg gatcacaatt atggaaagaa gttcctttga aaaaaatccc attgactttc
     4741 tggaagctaa aggatataag gaagttaaaa aagacctgat cattaaactg cctaaatata
     4801 gtctttttga gctggaaaac ggtaggaaac ggatgctggc tagtgccgga gaactgcaaa
     4861 aaggaaatga gctggctctg ccaagcaaat atgtgaattt tctgtatctg gctagtcatt
     4921 atgaaaagtt gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc
     4981 ataagcatta tctggatgag attattgagc aaatcagtga attttctaag agagttattc
     5041 tggcagatgc caatctggat aaagttctta gtgcatataa caaacataga gacaaaccaa
     5101 taagagaaca agcagaaaat atcattcatc tgtttacctt gaccaatctt ggagcacccg
     5161 ctgcttttaa atactttgat acaacaattg ataggaaaag atatacctct acaaaagaag
     5221 ttctggatgc cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt
     5281 tgagtcagct gggaggtgac cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc
     5341 ggaaagtgag tggtggagga agtggcgggt cagggtcgat ggacgcgaaa tcacttacgg
     5401 catggtcgag aacactggtt acgttcaagg acgtgtttgt ggactttaca cgtgaggagt
     5461 ggaaattgct ggatactgcg caacaaattg tgtatcgaaa tgtcatgctt gagaattaca
     5521 agaacctcgt cagtctcgga taccagttga cgaaaccgga tgtgatcctt aggctcgaaa
     5581 agggggaaga accttggctg gtatcgggag gtggttcggg tggctctgga tcaagcccaa
     5641 agaagaaacg gaaggtggaa gcctcagtgc aggtgaaaag ggtgctggaa aaatcccccg
     5701 gcaaactcct cgtgaagatg cccttccagg cttcccctgg cggaaaaggt gaagggggtg
     5761 gcgcaaccac atctgcccag gtcatggtca tcaagcgacc tggaaggaaa agaaaggccg
     5821 aggctgaccc tcaggccatt ccaaagaaac ggggacgcaa gccagggtcc gtggtcgcag
     5881 ctgcagcagc tgaggctaag aaaaaggcag tgaaggaaag ctccatccgc agtgtgcagg
     5941 agactgtcct gcccatcaag aagaggaaga ctagggagac cgtgtccatc gaggtcaaag
     6001 aagtggtcaa gcccctgctc gtgtccaccc tgggcgaaaa atctggaaag gggctcaaaa
     6061 catgcaagtc acctggacgg aaaagcaagg agtctagtcc aaaggggcgc tcaagctccg
     6121 cttctagtcc ccctaaaaag gaacaccatc accatcacca tcacgccgag tctcctaagg
     6181 ctcctatgcc actgctccca ccacctccac cacctgagcc acagtcaagc gaagacccca
     6241 tcagcccacc cgagcctcag gatctgtcct ctagtatttg caaagaggaa aagatgccca
     6301 gagcaggcag cctggagagt gatggctgtc caaaagaacc cgccaagacc cagcctatgg
     6361 tggcagccgc tgcaactacc accacaacca caactaccac agtggccgaa aaatacaagc
     6421 atcgcggcga gggcgaacga aaggacattg tgtcaagctc catgcccaga cctaaccggg
     6481 aggaaccagt cgatagtagg acacccgtga ctgagagagt ctcataaacg cgtacgcggc
     6541 cgctcgagca gaaactcatc tcagaagagg atctggcagc aaatgatatc ctggattaca
     6601 aggatgacga cgataaggtt taaacggccg gccgcggtca tagctgtttc ctgaacagat
     6661 cccgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact
     6721 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg
     6781 tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag
     6841 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct
     6901 tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag
     6961 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagagg
     7021 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca
     7081 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt
     7141 ctgattttaa aataactata ccagcaggag gacgtccaga cacagcatag gctacctggc
     7201 catgcccaac cggtgggaca tttgagttgc ttgcttggca ctgtcctctc atgcgttggg
     7261 tccactcagt agatgcctgt tgaattgggt acgcggccag cggcgagcgg tatcagctca
     7321 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtc
     7381 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     7441 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     7501 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     7561 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     7621 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     7681 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     7741 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     7801 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     7861 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     7921 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     7981 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     8041 gacgcgtaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     8101 gatccttttg cggccgcaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     8161 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     8221 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     8281 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     8341 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     8401 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     8461 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     8521 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     8581 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     8641 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     8701 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     8761 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     8821 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     8881 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     8941 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     9001 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     9061 attgtctcat gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac
     9121 gggccagagc tgccaggaaa cagctatgac catgtaatac gactcactat aggggatatc
     9181 agctggatgg cagttaac
//