LOCUS       sequence-343927-        5074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CasMINI-V3.1 in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5074)
  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 5074)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell. 
            2021 Aug 26. pii: S1097-2765(21)00648-1. doi: 
            10.1016/j.molcel.2021.08.008."
COMMENT     Sequence Label: pSLQ9824: CMV-SV40_NLS-CasMINI-V3.1--3xFlag-
            c...
FEATURES             Location/Qualifiers
     source          1..5074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             646..666
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2266..2331
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2347..2373
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     primer_bind     complement(2396..2413)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2402..2626
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2697..2713)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2697..2713)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2710..2732)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2721..2737
                     /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(join(2745..2751,2752..2769,2770..2775))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2790..2811
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2928..2945)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3099..3687)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3179..3198)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3858..4649,4650..4718))
                     /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     4481..4500
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4719..4823)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4902..4921)
                     /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 tgggacccaa gaaaaaacgc
      661 aaggtgggaa gcggatccgc caaaaacacc attaccaaaa cactgaaact gcgtattgtg
      721 cgtccgtata atagcgcaga agtggaaaaa attgttgccg acgaaaaaaa caaccgcgaa
      781 aaaatcgcac tggaaaagaa caaagacaaa gtgaaagaag cctgcagcaa acatctgaaa
      841 gttgcagcat attgtaccac acaggttgaa cgtaatgcat gcctgttttg taaagcacgt
      901 aaactggatg acaaattcta ccaaaaactg cgtggtcagt ttccggatgc agttttttgg
      961 caagaaatca gcgaaatttt tcgccagctg cagaaacagg cagcagaaat ctataatcag
     1021 agcctgatcg aactgtacta cgagattttt atcaaaggca aaggtattgc aaatgccagc
     1081 agcgttgaac attatctgag tagagtttgt tatagacgtg cagcagctct gtttaaaaac
     1141 gcagcaattg caagcggtct gcgtagcaaa atcaaaagca attttcgtct gaaagaactg
     1201 aaaaacatga aaagtggtct gccgaccacc aaaagcgata attttccgat tccgctggtt
     1261 aaacagaaag gtggtcagta taccggtttt gaaattagca atcataatag cgacttcatc
     1321 atcaagattc cgtttggtcg ttggcaggtc aaaaaagaga ttgataaata tcgtccgtgg
     1381 gagaaatttg actttgaaca ggttcagaaa agcccgaaac cgattagcct gctgctgagc
     1441 acccagcgtc gtaaacgtaa taaaggttgg agcaaagatg aaggcaccga agccgaaatc
     1501 aaaaaagtta tgaatggcga ttatcagacc agctacattg aagttaaacg tggcagcaaa
     1561 atctgtgaaa aaagcgcatg gatgctgaat ctgagcattg atgttccgaa aattgataaa
     1621 ggtgtggatc cgagcattat tggtggtatt gatgttggtg ttaaatcacc gctggtttgc
     1681 gcaattaaca atgcatttag ccgttatagc atcagcgata acgacctgtt tcacttcaac
     1741 aagaaaatgt ttgcacgtcg tcgtatcctg ctgaaaaaaa accgtcataa acgtgcaggt
     1801 catggtgcaa aaaacaaact gaaaccgatc accattctga ccgaaaaaag tgaacgtttt
     1861 cgcaaaaagc tgattgaacg ttgggcatgt gaaatcgcgg atttcttcat taaaaacaaa
     1921 gttggcaccg tgcagatgga aaatctggaa agcatgaaac gtaaagagga cagctatttt
     1981 aacattcgcc tgcgtggctt ttggccgtat gcagaaatgc agaacaaaat cgaattcaaa
     2041 ctgaagcagt atggcatcga aattcgtaaa gttgcaccga ataataccag caaaacctgt
     2101 agcaaatgtg gccatctgaa caactatttc aacttcgagt accgcaagaa aaacaaattc
     2161 ccgcacttta aatgcgaaaa atgcaacttc aaagaaaacg ccgattataa tgcagccctg
     2221 aatatttcaa acccgaaact gaaaagcacc aaagaggaac cggccgacta taaggaccac
     2281 gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gagcctgggc
     2341 agcggccctg ctgccaaacg cgttaaacta gactgagttt aaacccgctg atcagcctcg
     2401 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2461 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2521 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2581 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     2641 agaaccagct ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca
     2701 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     2761 agcataaagt gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     2821 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     2881 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     2941 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3001 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3061 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3121 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3181 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3241 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3301 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3361 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3421 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3481 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     3541 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3601 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3661 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3721 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3781 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3841 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3901 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3961 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4021 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4081 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4141 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4201 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4261 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4321 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4381 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4441 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4501 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4561 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4621 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4681 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4741 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4801 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     4861 tcgggagatc gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc
     4921 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     4981 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     5041 agggttaggc gttttgcgct gcttcgcgat gtac
//
