LOCUS       sequence-232190-        5800 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 5800)
  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 5800)
  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..5800
                     /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             2453..2506
                     /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(2640..2658)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    3248..3369
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3285..3304)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3339..3358
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3412..3428)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3412..3428)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3425..3447)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3436..3452
                     /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(3460..3466,3467..3484,3485..3490))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3505..3526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3643..3660)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3814..4402)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3894..3913)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4573..5364,5365..5433))
                     /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     5196..5215
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5434..5538)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5617..5636)
                     /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 cggatccagg tccggcggcg gagagggcag
     2461 aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc ggcccaatgg tgagcaaggg
     2521 cgagcgcgtg atcaaggagt tcatgcgctt caaggtgcac atggagggca gcatgaacgg
     2581 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     2641 caagctgaag gtgaccaagg gcggccccct gcccttcagc tgggacatcc tgagccccca
     2701 gttcatgtac ggcagccgcg ccttcaccaa gcaccccgcc gacatccccg actactacaa
     2761 gcagagcttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgc
     2821 cgtgaccgtg acccaggaca ccagcctgga ggacggcacc ctgatctaca aggtgaagct
     2881 gcgcggcacc aacttccccc ccgacggccc cgtgatgcag aagaagacca tgggctggga
     2941 ggccagcacc gagcgcctgt accccgagga cggcgtgctg aagggcgaca tcaagatggc
     3001 cctgcgcctg aaggacggcg gccgctacct ggccgacttc aagaccacct acaaggccaa
     3061 gaagcccgtg cagatgcccg gcgcctacaa cgtggaccgc aagctggaca tcaccagcca
     3121 caacgaggac tacaccgtgg tggagcagta cgagcgcagc gagggccgcc acagcaccgg
     3181 cggcatggac gagctgtaca agtaactgca gcgcggggat ctcatgctgg agttcttcgc
     3241 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     3301 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     3361 tgtatcttat taattaacgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     3421 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     3481 aaagtgtaaa gcctagggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3541 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3601 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3661 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3721 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3781 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3841 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3901 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3961 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4021 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4081 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4141 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4201 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4261 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4321 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4381 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4441 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4501 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4561 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4621 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4681 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4741 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4801 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4861 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4921 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4981 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5041 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5101 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5161 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5221 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5281 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5341 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5401 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5461 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5521 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     5581 agatcgatct cccgatcccc tagggtcgac tctcagtaca atctgctctg atgccgcata
     5641 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     5701 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     5761 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
//
