LOCUS       sequence-235385-        5752 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Mammalian expression, Transcription regulation module.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5752)
  AUTHORS   Teng F, Cui T, Feng G, Guo L, Xu K, Gao Q, Li T, Li J, Zhou Q, Li W
  TITLE     Repurposing CRISPR-Cas12b for mammalian genome engineering.
  JOURNAL   Cell Discov. 2018 Nov 27;4:63. doi: 10.1038/s41421-018-0069-3. 
            eCollection 2018.
  PUBMED    30510770
REFERENCE   2  (bases 1 to 5752)
  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: "Cell 
            Discov."; date: "2018-11-27"; volume: "4:63. doi"; pages: " 
            10.1038/s41421-018-0069-3. eCollection 2018"
COMMENT     Sequence Label: pCAG-MCP-VP64-2AmScarlet
FEATURES             Location/Qualifiers
     source          1..5752
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        77..456
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          683..1699
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1623..1645
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1707..1726
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1760..1778
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1790..1810
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1820..2206
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             2246..2266
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2282..2431
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             2432..2452
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2474..2527
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(2661..2679)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    3269..3390
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3306..3325)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3360..3379
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3433..3449)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3433..3449)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3446..3468)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3457..3473
                     /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(3481..3487,3488..3505,3506..3511))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3526..3547
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3664..3681)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3835..4423)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3915..3934)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4594..5385,5386..5454))
                     /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     5217..5236
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5455..5559)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5638..5657)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
       61 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
      121 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      181 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      241 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      301 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      361 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      421 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
      481 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      541 ttattttgtg cagcgatggg ggcggggggg gggggggaga ggtgcggcgg cagccaatca
      601 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
      661 aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc
      721 cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag
      781 cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt
      841 ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg
      901 gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc
      961 gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt
     1021 gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa
     1081 caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt
     1141 cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg
     1201 tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca
     1261 ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg
     1321 cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt
     1381 atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa
     1441 atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg
     1501 caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc
     1561 tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc
     1621 ggggttcggc ttctggcgtg tgaccggcgg ctctagcgcc tctgctaacc atgttcatgc
     1681 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt
     1741 tggcaaagct agtgaattct aatacgactc actataggcc gccaccatgc ccaagaagaa
     1801 gaggaaggtt cccgggatgg cttcaaactt tactcagttc gtgctcgtgg acaatggtgg
     1861 gacaggggat gtgacagtgg ctccttctaa tttcgctaat ggggtggcag agtggatcag
     1921 ctccaactca cggagccagg cctacaaggt gacatgcagc gtcaggcagt ctagtgccca
     1981 gaagagaaag tataccatca aggtggaggt ccccaaagtg gctacccaga cagtgggcgg
     2041 agtcgaactg cctgtcgccg cttggaggtc ctacctgaac atggagctca ctatcccaat
     2101 tttcgctacc aattctgact gtgaactcat cgtgaaggca atgcaggggc tcctcaaaga
     2161 cggtaatcct atcccttccg ccatcgccgc taactcaggt atctacagcg ctggaggagg
     2221 aggaagcgga ggaggaggta gcggacctaa gaaaaagagg aaggtggcgg ccgctcttaa
     2281 ggacgcattg gacgattttg atctggatat gctgggaagt gacgccctcg atgattttga
     2341 ccttgacatg cttggttcgg atgcccttga tgactttgac ctcgacatgc tcggcagtga
     2401 cgcccttgat gatttcgacc tggacatgct gccaaagaag aagaggaaag tcggatccag
     2461 gtccggcggc ggagagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc
     2521 cggcccaatg gtgagcaagg gcgagcgcgt gatcaaggag ttcatgcgct tcaaggtgca
     2581 catggagggc agcatgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     2641 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcag
     2701 ctgggacatc ctgagccccc agttcatgta cggcagccgc gccttcacca agcaccccgc
     2761 cgacatcccc gactactaca agcagagctt ccccgagggc ttcaagtggg agcgcgtgat
     2821 gaacttcgag gacggcggcg ccgtgaccgt gacccaggac accagcctgg aggacggcac
     2881 cctgatctac aaggtgaagc tgcgcggcac caacttcccc cccgacggcc ccgtgatgca
     2941 gaagaagacc atgggctggg aggccagcac cgagcgcctg taccccgagg acggcgtgct
     3001 gaagggcgac atcaagatgg ccctgcgcct gaaggacggc ggccgctacc tggccgactt
     3061 caagaccacc tacaaggcca agaagcccgt gcagatgccc ggcgcctaca acgtggaccg
     3121 caagctggac atcaccagcc acaacgagga ctacaccgtg gtggagcagt acgagcgcag
     3181 cgagggccgc cacagcaccg gcggcatgga cgagctgtac aagtaactgc agcgcgggga
     3241 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     3301 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     3361 tggtttgtcc aaactcatca atgtatctta ttaattaacg tcgacctcta gctagagctt
     3421 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3481 caacatacga gccggaagca taaagtgtaa agcctagggt gcctaatgag tgagctaact
     3541 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3601 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3661 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3721 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3781 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3841 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3901 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3961 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4021 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4081 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4141 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4201 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4261 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4321 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4381 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4441 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4501 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4561 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4621 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4681 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4741 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4801 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4861 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4921 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4981 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     5041 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     5101 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     5161 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     5221 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5281 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5341 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5401 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5461 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5521 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5581 acctgacgtc gacggatcgg gagatcgatc tcccgatccc ctagggtcga ctctcagtac
     5641 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     5701 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga cc
//
