LOCUS       sequence-120197-        5499 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Transient expression of humanized MS2 (V75E/A81G), fused to the VP64
            transcrption activator, in mammalian cells, under a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5499)
  AUTHORS   Shechner DM, Hacisuleyman E, Younger ST, Rinn JL
  TITLE     Multiplexable, locus-specific targeting of long RNAs with 
            CRISPR-Display.
  JOURNAL   Nat Methods. 2015 Jul;12(7):664-70. doi: 10.1038/nmeth.3433. Epub 
            2015 Jun 1.
  PUBMED    26030444
REFERENCE   2  (bases 1 to 5499)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nmeth.3433"; journalName: "Nat Methods"; date: "2015-07"; 
            volume: "12"; issue: "7"; pages: "664-70"
COMMENT     Sequence Label: pCMV_mCherry_2A_HA_2xMS2-VP64
FEATURES             Location/Qualifiers
     source          1..5499
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /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     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1124..1552
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1211..1230)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1421..1442
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1561..1581)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     enhancer        1582..1960
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1961..2164
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2115..2135
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2225..2234
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2237..2257
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2258..2965
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGREFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2403..2421)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2671..2690
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2865..2888
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2990..3043
                     /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"
     CDS             3050..3076
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             3050..3076
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3896..3916
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3941..4090
                     /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"
     primer_bind     complement(4167..4186)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4221..4240
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4300..4316)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4300..4316)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4313..4335)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4324..4340
                     /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(4348..4354,4355..4372,4373..4378))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4393..4414
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    4529..4620
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(4561..4580)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    4634..4682
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(4744..5329)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4824..4843)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
     1021 cccctatggt gcactctcag tacaatctgg catgctgggg atgcggtggg ctctatggct
     1081 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     1141 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1201 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1261 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1321 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1381 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1441 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1501 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     1561 ggaatgtgtg tcagttaggg tacattgatt attgactagt tattaatagt aatcaattac
     1621 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
     1681 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     1741 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     1801 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     1861 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     1921 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     1981 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     2041 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     2101 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     2161 agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatag acccaagctg
     2221 gctagccacc atggctccaa agaagaagcg taaggtaatg gtgagcaagg gcgaggagga
     2281 caacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa
     2341 cggccgcgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac
     2401 cgccaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca tcctgtcccc
     2461 tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt
     2521 gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg
     2581 cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa
     2641 gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg
     2701 ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca
     2761 gaggctgaag ctgaaggacg gcggccacta cgacgccgag gtcaagacca cctacaaggc
     2821 caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagctgg acatcacctc
     2881 ccacaacgag gactacacca tcgtggaaca gtacgagcgc gccgagggcc gccactccac
     2941 cggcggcatg gacgagctgt acaagggcag caccagcggc ggcagtggag agggcagagg
     3001 aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccaggcagtt acccctacga
     3061 cgtgcccgac tacgccggat ccggcgccgg taccagcaat ttcactcaat ttgtgctcgt
     3121 ggataacgga ggaacgggcg acgtgaccgt ggcaccaagc aactttgcta atggagtggc
     3181 cgagtggatt agctcaaata gcaggtccca ggcttacaag gtcacctgta gtgtccggca
     3241 atcatccgcc cagaatcgga aatatactat taaggtagaa gtgccgaaag tggcaactca
     3301 aaccgtgggg ggcgaggaat tgcctgtggc cggctggaga agctacctca acatggaact
     3361 caccattcca atcttcgcca caaacagcga ctgtgagctc atcgtcaaag ccatgcaggg
     3421 tctgctgaag gacggcaatc ctattccatc tgcaatcgcg gccaattccg gcatttatgg
     3481 ttccacgagc gggtcaggga gcaatttcac tcaatttgtg ctcgtggata acggaggaac
     3541 gggcgacgtg accgtggcac caagcaactt tgctaatgga gtggccgagt ggattagctc
     3601 aaatagcagg tcccaggctt acaaggtcac ctgtagtgtc cggcaatcat ccgcccagaa
     3661 tcggaaatat actattaagg tagaagtgcc gaaagtggca actcaaaccg tggggggcga
     3721 ggaattgcct gtggccggct ggagaagcta cctcaacatg gaactcacca ttccaatctt
     3781 cgccacaaac agcgactgtg agctcatcgt caaagccatg cagggtctgc tgaaggacgg
     3841 caatcctatt ccatctgcaa tcgcggccaa ttccggcatt tatggttccg gggaccccaa
     3901 gaagaagaga aaggtggagg ccagcggttc cggacgggct gacgcattgg acgattttga
     3961 tctggatatg ctgggaagtg acgccctcga tgattttgac cttgacatgc ttggttcgga
     4021 tgcccttgat gactttgacc tcgacatgct cggcagtgac gcccttgatg atttcgacct
     4081 ggacatgctg attaactgaa taaatgaggg cccgtttaaa cccgctgatc agttgtttat
     4141 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4201 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4261 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4321 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4381 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4441 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4501 cggtttgcgt attgggcgct cttccgctag agaaatgttc tggcacctgc acttgcactg
     4561 gggacagcct attttgctag tttgttttgt ttcgttttgt tttgatggag agcgtatgtt
     4621 agtactatcg attcacacaa aaaaccaaca cacagatgta atgaaaataa agatatttta
     4681 ttacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4741 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4801 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4861 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4921 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4981 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5041 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5101 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5161 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5221 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5281 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5341 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5401 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5461 atcaatctaa agtatatatg agtaaacttg gtctgacag
//
