LOCUS       Exported                6406 bp ds-DNA     circular SYN 01-OCT-2021
DEFINITION  Expression of TadA-TadA* (8e)-dCasMINI-V4 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pSLQ9757: CMV-BPNLS_TadA-linkerA-TadA*(8e)-Linker B-SV40
            NLS-d...
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6406)
  AUTHORS   Xu X, Chemparathy A, Zeng L, Kempton HR, Shang S, Nakamura M, Qi LS
  TITLE     Engineered miniature CRISPR-Cas system for mammalian genome 
            regulation and editing.
  JOURNAL   Mol Cell. 2021 Aug 26. pii: S1097-2765(21)00648-1. doi: 
            10.1016/j.molcel.2021.08.008.
  PUBMED    34480847
REFERENCE   2  (bases 1 to 6406)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 1, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6406
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        19..398
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        399..602
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     553..573
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      634..643
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             679..699
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1888..1908
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3508..3534
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3613..3639
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             3640..3705
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3728..3745)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3734..3958
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4029..4045)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4029..4045)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4042..4064)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4053..4069
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(4077..4107)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4122..4143
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4260..4277)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4431..5019)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4511..4530)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5190..6050)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5813..5832
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6051..6155)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6234..6253)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
       61 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
      121 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
      181 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
      241 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
      301 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
      361 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
      421 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
      481 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
      541 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
      601 ctggtttagt gaaccgtcag atccgctagc cgggccacca tgggaaaacg gacagccgac
      661 ggaagcgagt tcgagtcacc aaagaagaag cggaaagtct ctgaagtcga gtttagccac
      721 gagtattgga tgaggcacgc actgaccctg gcaaagcgag catgggatga aagagaagtc
      781 cccgtgggcg ccgtgctggt gcacaacaat agagtgatcg gagagggatg gaacaggcca
      841 atcggccgcc acgaccctac cgcacacgca gagatcatgg cactgaggca gggaggcctg
      901 gtcatgcaga attaccgcct gatcgatgcc accctgtatg tgacactgga gccatgcgtg
      961 atgtgcgcag gagcaatgat ccacagcagg atcggaagag tggtgttcgg agcacgggac
     1021 gccaagaccg gcgcagcagg ctccctgatg gatgtgctgc accaccccgg catgaaccac
     1081 cgggtggaga tcacagaggg aatcctggca gacgagtgcg ccgccctgct gagcgatttc
     1141 tttagaatgc ggagacagga gatcaaggcc cagaagaagg cacagagctc caccgactct
     1201 ggaggatcta gcggaggatc ctctggaagc gagacaccag gcacaagcga gtccgccaca
     1261 ccagagagct ccggcggctc ctccggagga tcctctgagg tggagttttc ccacgagtac
     1321 tggatgagac atgccctgac cctggccaag agggcacggg atgagaggga ggtgcctgtg
     1381 ggagccgtgc tggtgctgaa caatagagtg atcggcgagg gctggaacag agccatcggc
     1441 ctgcacgacc caacagccca tgccgaaatt atggccctga gacagggcgg cctggtcatg
     1501 cagaactaca gactgattga cgccaccctg tacgtgacat tcgagccttg cgtgatgtgc
     1561 gccggcgcca tgatccactc taggatcggc cgcgtggtgt ttggcgtgag gaactcaaaa
     1621 agaggcgccg caggctccct gatgaacgtg ctgaactacc ccggcatgaa tcaccgcgtc
     1681 gaaattaccg agggaatcct ggcagatgaa tgtgccgccc tgctgtgcga tttctatcgg
     1741 atgcctagac aggtgttcaa tgctcagaag aaggcccaga gctccatcaa ctctggagga
     1801 tctagcggag gatcctctgg cagcgagaca ccaggaacaa gcgagtcagc aacaccagag
     1861 agcagtggcg gcagcagcgg cggcagcccc aagaaaaaac gcaaggtggg aagcggatcc
     1921 gccaaaaaca ccattaccaa aacactgaaa ctgcgtattg tgcgtccgta taatagcgca
     1981 gaagtggaaa aaattgttgc cgacgaaaaa aacaaccgcg aaaaaatcgc actggaaaag
     2041 aacaaagaca aagtgaaaga agcctgcagc aaacatctga aagttgcagc atattgtacc
     2101 acacaggttg aacgtaatgc atgcctgttt tgtaaagcac gtaaactgga tgacaaattc
     2161 taccaaaaac tgcgtggtca gtttccggat gcagtttttt ggcaagaaat cagcgaaatt
     2221 tttcgccagc tgcagaaaca ggcagcagaa atctataatc agagcctgat cgaactgtac
     2281 tacgagattt ttatcaaagg caaaggtatt gcaaatgcca gcagcgttga acattatctg
     2341 agtagagttt gttatagacg tgcagcagaa ctgtttaaaa acgcagcaat tgcaagcggt
     2401 ctgcgtagca aaatcaaaag caattttcgt ctgaaagaac tgaaaaacat gaaaagtggt
     2461 ctgccgacca ccaaaagcga taattttccg attccgctgg ttaaacagaa aggtggtcag
     2521 tataccggtt ttgaaattag caatcataat agcgacttca tcatcaagat tccgtttggt
     2581 cgttggcagg tcaaaaaaga gattgataaa tatcgtccgt gggagaaatt tgactttgaa
     2641 caggttcaga aaagcccgaa accgattagc ctgctgctga gcacccagcg tcgtaaacgt
     2701 aataaaggtt ggagcaaaga tgaaggcacc gaagccgaaa tcaaaaaagt tatgaatggc
     2761 gattatcaga ccagctacat tgaagttaaa cgtggcagca aaatctgtga aaaaagcgca
     2821 tggatgctga atctgagcat tgatgttccg aaaattgata aaggtgtgga tccgagcatt
     2881 attggtggta ttgcagttgg tgttagatca ccgctggttt gcgcaattaa caatgcattt
     2941 agccgttata gcatcagcga taacgacctg tttcacttca acaagaaaat gtttgcacgt
     3001 cgtcgtatcc tgctgaaaaa aaaccgtcat aaacgtgcag gtcatggtgc aaaaaacaaa
     3061 ctgaaaccga tcaccattct gaccgaaaaa agtgaacgtt ttcgcaaaaa gctgattgaa
     3121 cgttgggcat gtgaaatcgc ggatttcttc attaaaaaca aagttggcac cgtgcagatg
     3181 gaaaatctgg aaagcatgaa acgtaaagag gacagctatt ttaacattcg cctgcgtggc
     3241 ttttggccgt atgcagaaat gcagaacaaa atcgaattca aactgaagca gtatggcatc
     3301 gaaattcgta aagttgcacc gaataatacc agcaaaacct gtagcaaatg tggccatctg
     3361 aacaactatt tcaacttcga gtaccgcaag aaaaacaaat tcccgcactt taaatgcgaa
     3421 aaatgcaact tcaaagaaaa cgccgcgtat aatgcagccc tgaatatttc aaacccgaaa
     3481 ctgaaaagca ccaaagagag accggcctat ccctatgacg tgcccgatta tgccagcctg
     3541 ggcagcggcg acggcattgg tagtgggagc aacggcagca gcctccatat gggtggaggc
     3601 tccggggaag atcctgctgc caaacgcgtt aaactagacg actataagga ccacgacgga
     3661 gactacaagg atcatgatat tgattacaaa gacgatgacg ataagtgagt ttaaacccgc
     3721 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3781 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3841 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3901 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3961 tctgaggcgg aaagaaccag ctggggctcg ataccgtcga cctctagcta gagcttggcg
     4021 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4081 atacgagccg gaagcataaa gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca
     4141 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4201 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4261 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4321 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4381 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4441 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4501 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4561 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4621 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4681 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4741 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4801 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4861 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4921 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4981 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5041 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5101 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5161 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5221 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5281 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5341 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5401 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5461 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5521 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5581 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5641 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5701 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5761 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5821 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5881 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5941 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6001 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6061 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6121 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6181 gacgtcgacg gatcgggaga tcgatctccc gatcccctag ggtcgactct cagtacaatc
     6241 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     6301 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
     6361 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtac
//
