LOCUS       sequence-383750-        7870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 24 Broccoli-tagged beta-actin mRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7870)
  AUTHORS   Li X, Kim H, Litke JL, Wu J, Jaffrey SR
  TITLE     Fluorophore-Promoted RNA Folding and Photostability Enables Imaging 
            of Single Broccoli-Tagged mRNAs in Live Mammalian Cells.
  JOURNAL   Angew Chem Int Ed Engl. 2020 Mar 9;59(11):4511-4518. doi: 
            10.1002/anie.201914576. Epub 2020 Jan 28.
  PUBMED    31850609
REFERENCE   2  (bases 1 to 7870)
  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"; doi: 
            "10.1002/anie.201914576"; journalName: "Angew Chem Int Ed Engl"; 
            date: "2020-03-9- 9"; volume: "59"; issue: "11"; pages: "4511-4518"
COMMENT     Sequence Label: CMV-mCherry-3'UTR of beta-actin-(F30-2xdBroccoli)6
FEATURES             Location/Qualifiers
     source          1..7870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             898..1608
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYRAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1043..1061)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1311..1330
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(3424..3442)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3425..3442)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3462..3479)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3468..3692
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3738..4166
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3825..3844)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4035..4056
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4175..4195)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4180..4509
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4360..4495
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4422..4441
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4576..5370
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4630..4649)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5240..5259
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5544..5665
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5581..5600)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5635..5654
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5714..5730)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5714..5730)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5727..5749)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5738..5754
                     /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(5762..5768,5769..5786,5787..5792))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5807..5828
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5945..5962)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6116..6703)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6196..6215)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6874..7665,7666..7734))
                     /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     7497..7516
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7735..7839)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttaccatg
      901 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
      961 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     1021 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     1081 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     1141 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     1201 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     1261 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     1321 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     1381 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     1441 gtcaagacca cctacagggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     1501 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     1561 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaagg atcttagtca
     1621 ttccaaatat gagatgcgtt gttacaggaa gtcccttgcc atcctaaaag ccaccccact
     1681 tctctctaag gagaatggcc cagtcctctc ccaagtccac acaggggagg tgatagcatt
     1741 gctttcgtgt aaattatgta atgcaaaatt tttttaatct tcgccttaat acttttttat
     1801 tttgttttat tttgaatgat gagccttcgt gccccccctt cccccttttt tgtcccccaa
     1861 cttgagatgt atgaaggctt ttggtctccc tgggagtggg tggaggcagc cagggcttac
     1921 ctgtacactg acttgagacc agttgattac gaattcggat cctcgccttg tgtctgttgg
     1981 agacggtcgg gtccaaatga gacggtcggg tccagaagtt cgcttctgtc gagtagagtg
     2041 tgggctcatt tgtcgagtag agtgtgggct ccaacagact ctgacgatcc agacggtcgg
     2101 gtccatctga gacggtcggg tccagtagtt cgctactgtc gagtagagtg tgggctcaga
     2161 tgtcgagtag agtgtgggct ggatcgttca aggcgaatat tactactcgc gatgtctatg
     2221 cgggagacgg tcgggtccac ctgagacggt cgggtccact gattcgtcag tgtcgagtag
     2281 agtgtgggct caggtgtcga gtagagtgtg ggctcccgca tagtctgatc atccagacgg
     2341 tcgggtccat ttgagacggt cgggtccaga agttcgcttc tgtcgagtag agtgtgggct
     2401 caaatgtcga gtagagtgtg ggctggatga ttcatcgcga agatcctcgc cttgtgtctg
     2461 ttggagacgg tcgggtccaa atgagacggt cgggtccaga agttcgcttc tgtcgagtag
     2521 agtgtgggct catttgtcga gtagagtgtg ggctccaaca gactctgacg atccagacgg
     2581 tcgggtccat ctgagacggt cgggtccagt agttcgctac tgtcgagtag agtgtgggct
     2641 cagatgtcga gtagagtgtg ggctggatcg ttcaaggcga atattactac tcgcgatgtc
     2701 tatgcgggag acggtcgggt ccacctgaga cggtcgggtc cactgattcg tcagtgtcga
     2761 gtagagtgtg ggctcaggtg tcgagtagag tgtgggctcc cgcatagtct gatcatccag
     2821 acggtcgggt ccatttgaga cggtcgggtc cagaagttcg cttctgtcga gtagagtgtg
     2881 ggctcaaatg tcgagtagag tgtgggctgg atgattcatc gcgaagatcc tcgccttgtg
     2941 tctgttggag acggtcgggt ccaaatgaga cggtcgggtc cagaagttcg cttctgtcga
     3001 gtagagtgtg ggctcatttg tcgagtagag tgtgggctcc aacagactct gacgatccag
     3061 acggtcgggt ccatctgaga cggtcgggtc cagtagttcg ctactgtcga gtagagtgtg
     3121 ggctcagatg tcgagtagag tgtgggctgg atcgttcaag gcgaatatta ctactcgcga
     3181 tgtctatgcg ggagacggtc gggtccacct gagacggtcg ggtccactga ttcgtcagtg
     3241 tcgagtagag tgtgggctca ggtgtcgagt agagtgtggg ctcccgcata gtctgatcat
     3301 ccagacggtc gggtccattt gagacggtcg ggtccagaag ttcgcttctg tcgagtagag
     3361 tgtgggctca aatgtcgagt agagtgtggg ctggatgatt catcgcgaag atctgggccc
     3421 tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg tgccttctag
     3481 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3541 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3601 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     3661 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     3721 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3781 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3841 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3901 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3961 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4021 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4081 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4141 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     4201 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4261 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4321 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     4381 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4441 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     4501 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     4561 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     4621 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     4681 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     4741 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     4801 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     4861 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     4921 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     4981 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     5041 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     5101 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     5161 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     5221 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     5281 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     5341 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     5401 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     5461 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5521 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     5581 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5641 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     5701 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5761 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5821 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5881 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5941 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6001 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     6061 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6121 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6181 acccgacagg actataaaga taccaggcgt ttcccctgga agctccctcg tgcgctctcc
     6241 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6301 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6361 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6421 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6481 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6541 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6601 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6661 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6721 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6781 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6841 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6901 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6961 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     7021 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7081 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7141 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     7201 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7261 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7321 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7381 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7441 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7501 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7561 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7621 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7681 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7741 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7801 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     7861 acctgacgtc
//
