LOCUS       sequence-459530-        6765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express circular RNA translation reporter with 
            1xALFATag-5xSunTag_codon_optimized-8xAAA under CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6765)
  AUTHORS   Madern MF, Yang S, Witteveen O, Segeren HA, Bauer M, Tanenbaum ME
  TITLE     Long-term imaging of individual ribosomes reveals ribosome 
            cooperativity in mRNA translation.
  JOURNAL   Cell. 2025 Jan 23:S0092-8674(25)00045-5. doi: 
            10.1016/j.cell.2025.01.016.
  PUBMED    39892379
REFERENCE   2  (bases 1 to 6765)
  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. 2025 
            Jan 23:S0092-8674(25)00045-5. doi: 10.1016/j.cell.2025.01.016."
COMMENT     Sequence Label: 
            pAV-CMVp-2xTetO-Tornado-1xALFATag-5xSunTag_codon_optimized+8xAAA
FEATURES             Location/Qualifiers
     source          1..6765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     150..169
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     enhancer        607..986
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        987..1190
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    1192..1210
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1213..1231
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1241..1265
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1455..1493
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             1518..1574
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1590..1646
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1662..1718
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1734..1790
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1806..1862
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1884..1904
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1932..1941
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2132..2253
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2302..2431
                     /label=AAV2 ITR
     rep_origin      complement(2525..2980)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2662..2683)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2874..2893
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3007..3111
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3112..3180,3181..3972)
                     /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     complement(3330..3349)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4143..4731
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4632..4651
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5000..5134
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5559..5579)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          5709..5774
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(join(6194..6765,1..223))
                     /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(6305..6324)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac
       61 cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg
      121 cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc
      181 aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc
      241 ctgtctcttg atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca
      301 tccagtttac tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg
      361 gttcgcttgc tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag
      421 ctacctgctt tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat
      481 tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc
      541 agcccttgcg ccctgagtgc ttgcggcagc gtgaagcttt ttgcaaaagc taggcctggg
      601 atccttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      661 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      721 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      781 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      841 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      901 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      961 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     1021 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     1081 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     1141 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca
     1201 gtgatagaga tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc
     1261 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
     1321 ccagcctccg gactctagcg tttaaacgcg tactgtcgac gggccgcact cgccggtccc
     1381 aagcccggat aaaatgggag ggggcgggaa accgcctaac cataccgagt gcggccgcag
     1441 gctccggatc tggctccaga ctggaggagg aactgagacg gagactgaca gaacctggag
     1501 gcggcggcag cactggcgaa gagctgctga gcaagaacta ccatctggaa aatgaggtag
     1561 cacgcctgaa aaagggaagc ggctccggcg aggagctgct gtccaagaac tatcatctgg
     1621 aaaacgaggt agcaaggctg aagaagggca gcggctccgg agaggaactg ctgtctaaga
     1681 actatcatct ggaaaacgaa gttgctcgcc tgaagaaggg atctggctcc ggagaagaac
     1741 tgctgtccaa gaactatcac ctggagaacg aagttgctcg cctgaaaaaa ggcagcggat
     1801 ccggagaaga actgctgagc aaaaattacc atctggagaa cgaagttgca cggctgaaga
     1861 aaggctccgg cagcggagga actcctaaaa agaaacgcaa ggttaataaa aaaaaaaaaa
     1921 aaaaaaaaaa agccaccatg gctagcgcgg tcggcgtgga ctgtagaaca ctgccaatgc
     1981 cggtcccaag cccggataaa agtggagggt acagtccacg ctctagagcg gacttcggtc
     2041 cgctttttac taggacctgc aggcatgcaa gcttgacgtc ggttaccgat atccatatgg
     2101 cgaccgcatc gatctcgagc cgaggactag taacttgttt attgcagctt ataatggtta
     2161 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2221 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc ttacgtagat aagtagcatg
     2281 gcgggttaat cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc
     2341 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     2401 gggcggcctc agtgagcgag cgagcgcgca gagagggaca gatctgcaga tctgctggcg
     2461 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     2521 atggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     2581 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     2641 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     2701 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     2761 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     2821 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     2881 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     2941 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     3001 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3061 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3121 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3181 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3241 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3301 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     3361 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3421 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3481 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3541 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3601 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     3661 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3721 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3781 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3841 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3901 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3961 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4021 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4081 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4141 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4201 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4261 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     4321 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4381 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4441 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4501 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4561 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4621 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4681 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4741 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4801 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4861 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg agcgcgcaga gagggagtgg
     4921 ccaactccat cactaggggt tcctggaggg gtggagtcgt gacgtgaatt acgtcatagg
     4981 gttagggagg tcctggatcg atccagacat gataagatac attgatgagt ttggacaaac
     5041 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     5101 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     5161 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     5221 tggtatggct gattatgatc tctagtcaag gcactataca tcaaatattc cttattaacc
     5281 cctttacaaa ttaaaaagct aaaggtacac aatttttgag catagttatt aatagcagac
     5341 actctatgcc tgtgtggagt aagaaaaaac agtatgttat gattataact gttatgccta
     5401 cttataaagg ttacagaata tttttccata attttcttgt atagcagtgc agctttttcc
     5461 tttgtggtgt aaatagcaaa gcaagcaaga gttctattac taaacacagc atgactcaaa
     5521 aaacttagca attctgaagg aaagtccttg gggtcttcta cctttctctt cttttttgga
     5581 ggagtagaat gttgagagtc agcagtagcc tcatcatcac tagatggcat ttcttctgag
     5641 caaaacaggt tttcctcatt aaaggcattc caccactgct cccattcatc agttccatag
     5701 gttggaatct aaaatacaca aacaattaga atcagtagtt taacacatta tacacttaaa
     5761 aattttatat ttaccttaga gctttaaatc tctgtaggta gtttgtccaa ttatgtcaca
     5821 ccacagaagt aaggttcctt cacaaagatc cggaccaaag cggccatcgt gcctccccac
     5881 tcctgcagtt cgggggcatg gatgcgcgga tagccgctgc tggtttcctg gatgccgacg
     5941 gatttgcact gccggtagaa ctccgcgagg tcgtccagcc tcaggcagca gctgaaccaa
     6001 ctcgcgaggg gatcgagccc ggggtgggcg aagaactcca gcatgagatc cccgcgctgg
     6061 aggatcatcc agccggcgtc ccggaaaacg attccgaagc ccaacctttc atagaaggcg
     6121 gcggtggaat cgaaatctcg tgatggcagg ttgggcgtcg cttggtcggt catttcgaac
     6181 cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
     6241 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt
     6301 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc
     6361 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat
     6421 cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca
     6481 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg
     6541 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg
     6601 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg
     6661 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt
     6721 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaa
//
