LOCUS       sequence-232191-        7213 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression, Transcription regulation module.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7213)
  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 7213)
  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..7213
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..665
                     /label=chicken beta-actin promoter
     intron          666..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1739..1757
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1769..1789
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1799..2185
                     /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             2225..2245
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2261..2410
                     /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             2411..2431
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2864..3220
                     /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             3239..3808
                     /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             3824..3844
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3866..3919
                     /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(4053..4071)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    4661..4782
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4698..4717)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4752..4771
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4825..4841)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4825..4841)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4838..4860)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4849..4865
                     /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(4873..4879,4880..4897,4898..4903))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4918..4939
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5056..5073)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5227..5815)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5307..5326)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5986..6777,6778..6846))
                     /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     6609..6628
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6847..6951)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7030..7049)
                     /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 gactatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatgggt cgaggtgagc cccacgttct gcttcactct
      421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      481 tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg
      541 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      661 gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg
      841 aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccat ctccagcctc ggggctgtcc
     1561 gcagggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagtgcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagcta gtgaattcta
     1741 atacgactca ctataggccg ccaccatgcc caagaagaag aggaaggttc ccgggatggc
     1801 ttcaaacttt actcagttcg tgctcgtgga caatggtggg acaggggatg tgacagtggc
     1861 tccttctaat ttcgctaatg gggtggcaga gtggatcagc tccaactcac ggagccaggc
     1921 ctacaaggtg acatgcagcg tcaggcagtc tagtgcccag aagagaaagt ataccatcaa
     1981 ggtggaggtc cccaaagtgg ctacccagac agtgggcgga gtcgaactgc ctgtcgccgc
     2041 ttggaggtcc tacctgaaca tggagctcac tatcccaatt ttcgctacca attctgactg
     2101 tgaactcatc gtgaaggcaa tgcaggggct cctcaaagac ggtaatccta tcccttccgc
     2161 catcgccgct aactcaggta tctacagcgc tggaggagga ggaagcggag gaggaggtag
     2221 cggacctaag aaaaagagga aggtggcggc cgctcttaag gacgcattgg acgattttga
     2281 tctggatatg ctgggaagtg acgccctcga tgattttgac cttgacatgc ttggttcgga
     2341 tgcccttgat gactttgacc tcgacatgct cggcagtgac gcccttgatg atttcgacct
     2401 ggacatgctg ccaaagaaga agaggaaagt caagcttagc cagtacctgc ccgacaccga
     2461 cgaccggcac cggatcgagg aaaagcggaa gcggacctac gagacattca agagcatcat
     2521 gaagaagtcc cccttcagcg gccccaccga ccctagacct ccacctagaa gaatcgccgt
     2581 gcccagcaga tccagcgcca gcgtgccaaa acctgccccc cagccttacc ccttcaccag
     2641 cagcctgagc accatcaact acgacgagtt ccctaccatg gtgttcccca gcggccagat
     2701 ctctcaggcc tctgctctgg ctccagcccc tcctcaggtg ctgcctcagg ctcctgctcc
     2761 tgcaccagct ccagccatgg tgtctgcact ggctcaggca ccagcacccg tgcctgtgct
     2821 ggctcctgga cctccacagg ctgtggctcc accagcccct aaacctacac aggccggcga
     2881 gggcacactg tctgaagctc tgctgcagct gcagttcgac gacgaggatc tgggagccct
     2941 gctgggaaac agcaccgatc ctgccgtgtt caccgacctg gccagcgtgg acaacagcga
     3001 gttccagcag ctgctgaacc agggcatccc tgtggcccct cacaccaccg agcccatgct
     3061 gatggaatac cccgaggcca tcacccggct cgtgacaggc gctcagaggc ctcctgatcc
     3121 agctcctgcc cctctgggag caccaggcct gcctaatgga ctgctgtctg gcgacgagga
     3181 cttcagctct atcgccgata tggatttctc agccttgctg ggctctggca gcggcagccg
     3241 ggattccagg gaagggatgt ttttgccgaa gcctgaggcc ggctccgcta ttagtgacgt
     3301 gtttgagggc cgcgaggtgt gccagccaaa acgaatccgg ccatttcatc ctccaggaag
     3361 tccatgggcc aaccgcccac tccccgccag cctcgcacca acaccaaccg gtccagtaca
     3421 tgagccagtc gggtcactga ccccggcacc agtccctcag ccactggatc cagcgcccgc
     3481 agtgactccc gaggccagtc acctgttgga ggatcccgat gaagagacga gccaggctgt
     3541 caaagccctt cgggagatgg ccgatactgt gattccccag aaggaagagg ctgcaatctg
     3601 tggccaaatg gacctttccc atccgccccc aaggggccat ctggatgagc tgacaaccac
     3661 acttgagtcc atgaccgagg atctgaacct ggactcaccc ctgaccccgg aattgaacga
     3721 gattctggat accttcctga acgacgagtg cctcttgcat gccatgcata tcagcacagg
     3781 actgtccatc ttcgacacat ctctgtttag gtccggcggc ggacccaaga agaagcgcaa
     3841 ggtgctcgag aggtccggcg gcggagaggg cagaggaagt cttctaacat gcggtgacgt
     3901 ggaggagaat cccggcccaa tggtgagcaa gggcgagcgc gtgatcaagg agttcatgcg
     3961 cttcaaggtg cacatggagg gcagcatgaa cggccacgag ttcgagatcg agggcgaggg
     4021 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc
     4081 cctgcccttc agctgggaca tcctgagccc ccagttcatg tacggcagcc gcgccttcac
     4141 caagcacccc gccgacatcc ccgactacta caagcagagc ttccccgagg gcttcaagtg
     4201 ggagcgcgtg atgaacttcg aggacggcgg cgccgtgacc gtgacccagg acaccagcct
     4261 ggaggacggc accctgatct acaaggtgaa gctgcgcggc accaacttcc cccccgacgg
     4321 ccccgtgatg cagaagaaga ccatgggctg ggaggccagc accgagcgcc tgtaccccga
     4381 ggacggcgtg ctgaagggcg acatcaagat ggccctgcgc ctgaaggacg gcggccgcta
     4441 cctggccgac ttcaagacca cctacaaggc caagaagccc gtgcagatgc ccggcgccta
     4501 caacgtggac cgcaagctgg acatcaccag ccacaacgag gactacaccg tggtggagca
     4561 gtacgagcgc agcgagggcc gccacagcac cggcggcatg gacgagctgt acaagtaact
     4621 gcagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     4681 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4741 gcattctagt tgtggtttgt ccaaactcat caatgtatct tattaattaa cgtcgacctc
     4801 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4861 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctagg gtgcctaatg
     4921 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4981 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5041 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5101 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5161 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5221 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5281 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5341 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5401 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5461 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5521 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5581 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5641 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5701 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5761 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5821 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5881 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5941 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6001 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6061 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6121 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6181 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6241 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6301 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6361 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6421 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6481 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6541 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6601 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6661 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6721 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6781 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6841 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6901 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6961 ccgaaaagtg ccacctgacg tcgacggatc gggagatcga tctcccgatc ccctagggtc
     7021 gactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     7081 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     7141 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
     7201 acgggccaga tat
//
