LOCUS       sequence-235383-        7217 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 7217)
  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 7217)
  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-VPR-2AmScarlet
FEATURES             Location/Qualifiers
     source          1..7217
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          666..1682
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1606..1628
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1690..1709
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1743..1761
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1773..1793
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1803..2189
                     /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             2229..2249
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2265..2414
                     /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             2415..2435
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2868..3224
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             3243..3812
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     CDS             3828..3848
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3870..3923
                     /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(4057..4075)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    4665..4786
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4702..4721)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4756..4775
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4829..4845)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4829..4845)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4842..4864)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4853..4869
                     /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(4877..4883,4884..4901,4902..4907))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4922..4943
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5060..5077)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5231..5819)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5311..5330)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5990..6781,6782..6850))
                     /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     6613..6632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6851..6955)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7034..7053)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
      421 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
      481 gcagcgatgg gggcgggggg ggggggggns gcgcgccrgg sggggsgggg sggggsgrgg
      541 ggsggggsgg ggsgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      661 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc
      721 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
      781 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc
      841 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg
      901 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg
      961 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg
     1021 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg
     1081 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc
     1141 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg
     1201 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg
     1261 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg
     1321 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga
     1381 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc
     1441 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg
     1501 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct
     1561 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1621 gtgtgaccgg cggctctagc gcctctgcta accatgttca tgccttcttc tttttcctac
     1681 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gctagtgaat
     1741 tctaatacga ctcactatag gccgccacca tgcccaagaa gaagaggaag gttcccggga
     1801 tggcttcaaa ctttactcag ttcgtgctcg tggacaatgg tgggacaggg gatgtgacag
     1861 tggctccttc taatttcgct aatggggtgg cagagtggat cagctccaac tcacggagcc
     1921 aggcctacaa ggtgacatgc agcgtcaggc agtctagtgc ccagaagaga aagtatacca
     1981 tcaaggtgga ggtccccaaa gtggctaccc agacagtggg cggagtcgaa ctgcctgtcg
     2041 ccgcttggag gtcctacctg aacatggagc tcactatccc aattttcgct accaattctg
     2101 actgtgaact catcgtgaag gcaatgcagg ggctcctcaa agacggtaat cctatccctt
     2161 ccgccatcgc cgctaactca ggtatctaca gcgctggagg aggaggaagc ggaggaggag
     2221 gtagcggacc taagaaaaag aggaaggtgg cggccgctct taaggacgca ttggacgatt
     2281 ttgatctgga tatgctggga agtgacgccc tcgatgattt tgaccttgac atgcttggtt
     2341 cggatgccct tgatgacttt gacctcgaca tgctcggcag tgacgccctt gatgatttcg
     2401 acctggacat gctgccaaag aagaagagga aagtcaagct tagccagtac ctgcccgaca
     2461 ccgacgaccg gcaccggatc gaggaaaagc ggaagcggac ctacgagaca ttcaagagca
     2521 tcatgaagaa gtcccccttc agcggcccca ccgaccctag acctccacct agaagaatcg
     2581 ccgtgcccag cagatccagc gccagcgtgc caaaacctgc cccccagcct taccccttca
     2641 ccagcagcct gagcaccatc aactacgacg agttccctac catggtgttc cccagcggcc
     2701 agatctctca ggcctctgct ctggctccag cccctcctca ggtgctgcct caggctcctg
     2761 ctcctgcacc agctccagcc atggtgtctg cactggctca ggcaccagca cccgtgcctg
     2821 tgctggctcc tggacctcca caggctgtgg ctccaccagc ccctaaacct acacaggccg
     2881 gcgagggcac actgtctgaa gctctgctgc agctgcagtt cgacgacgag gatctgggag
     2941 ccctgctggg aaacagcacc gatcctgccg tgttcaccga cctggccagc gtggacaaca
     3001 gcgagttcca gcagctgctg aaccagggca tccctgtggc ccctcacacc accgagccca
     3061 tgctgatgga ataccccgag gccatcaccc ggctcgtgac aggcgctcag aggcctcctg
     3121 atccagctcc tgcccctctg ggagcaccag gcctgcctaa tggactgctg tctggcgacg
     3181 aggacttcag ctctatcgcc gatatggatt tctcagcctt gctgggctct ggcagcggca
     3241 gccgggattc cagggaaggg atgtttttgc cgaagcctga ggccggctcc gctattagtg
     3301 acgtgtttga gggccgcgag gtgtgccagc caaaacgaat ccggccattt catcctccag
     3361 gaagtccatg ggccaaccgc ccactccccg ccagcctcgc accaacacca accggtccag
     3421 tacatgagcc agtcgggtca ctgaccccgg caccagtccc tcagccactg gatccagcgc
     3481 ccgcagtgac tcccgaggcc agtcacctgt tggaggatcc cgatgaagag acgagccagg
     3541 ctgtcaaagc ccttcgggag atggccgata ctgtgattcc ccagaaggaa gaggctgcaa
     3601 tctgtggcca aatggacctt tcccatccgc ccccaagggg ccatctggat gagctgacaa
     3661 ccacacttga gtccatgacc gaggatctga acctggactc acccctgacc ccggaattga
     3721 acgagattct ggataccttc ctgaacgacg agtgcctctt gcatgccatg catatcagca
     3781 caggactgtc catcttcgac acatctctgt ttaggtccgg cggcggaccc aagaagaagc
     3841 gcaaggtgct cgagaggtcc ggcggcggag agggcagagg aagtcttcta acatgcggtg
     3901 acgtggagga gaatcccggc ccaatggtga gcaagggcga gcgcgtgatc aaggagttca
     3961 tgcgcttcaa ggtgcacatg gagggcagca tgaacggcca cgagttcgag atcgagggcg
     4021 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg
     4081 gccccctgcc cttcagctgg gacatcctga gcccccagtt catgtacggc agccgcgcct
     4141 tcaccaagca ccccgccgac atccccgact actacaagca gagcttcccc gagggcttca
     4201 agtgggagcg cgtgatgaac ttcgaggacg gcggcgccgt gaccgtgacc caggacacca
     4261 gcctggagga cggcaccctg atctacaagg tgaagctgcg cggcaccaac ttcccccccg
     4321 acggccccgt gatgcagaag aagaccatgg gctgggaggc cagcaccgag cgcctgtacc
     4381 ccgaggacgg cgtgctgaag ggcgacatca agatggccct gcgcctgaag gacggcggcc
     4441 gctacctggc cgacttcaag accacctaca aggccaagaa gcccgtgcag atgcccggcg
     4501 cctacaacgt ggaccgcaag ctggacatca ccagccacaa cgaggactac accgtggtgg
     4561 agcagtacga gcgcagcgag ggccgccaca gcaccggcgg catggacgag ctgtacaagt
     4621 aactgcagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4681 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4741 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttattaa ttaacgtcga
     4801 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4861 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tagggtgcct
     4921 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4981 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5041 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5101 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5161 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5221 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5281 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5341 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5401 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5461 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5521 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5581 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5641 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5701 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5761 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5821 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5881 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5941 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6001 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6061 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6121 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6181 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6241 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6301 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6361 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6421 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6481 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6541 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6601 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6661 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6721 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6781 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6841 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6901 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6961 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcgatctccc gatcccctag
     7021 ggtcgactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     7081 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
     7141 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
     7201 atgtacgggc cagatat
//
