LOCUS       sequence-459533-        6807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express circular RNA translation reporter with 
            1xALFATag-5xSunTag_codon_optimized-4x(6xAAA) under CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6807)
  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 6807)
  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-4x(6xAAA)
FEATURES             Location/Qualifiers
     source          1..6807
                     /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             1470..1508
                     /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             1548..1604
                     /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             1620..1676
                     /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             1710..1766
                     /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             1782..1838
                     /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             1872..1928
                     /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             1950..1970
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1974..1983
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2174..2295
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2344..2473
                     /label=AAV2 ITR
     rep_origin      complement(2567..3022)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2704..2725)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2916..2935
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3049..3153
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3154..3222,3223..4014)
                     /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(3372..3391)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4185..4773
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4674..4693
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5042..5176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5601..5621)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          5751..5816
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(join(6236..6807,1..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(6347..6366)
                     /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 gcaaaaaaaa aaaaaaaaaa ggatctggct ccagactgga ggaggaactg agacggagac
     1501 tgacagaacc tggatctggc aaaaaaaaaa aaaaaaaagg cggcagcgaa gagctgctga
     1561 gcaagaacta ccatctggaa aatgaggtag cacgcctgaa aaagggaagc ggctccggcg
     1621 aggagctgct gtccaagaac tatcatctgg aaaacgaggt agcaaggctg aagaagggca
     1681 gcggcaaaaa aaaaaaaaaa aaatccggag aggaactgct gtctaagaac tatcatctgg
     1741 aaaacgaagt tgctcgcctg aagaagggat ctggctccgg agaagaactg ctgtccaaga
     1801 actatcacct ggagaacgaa gttgctcgcc tgaaaaaagg cagcggaaaa aaaaaaaaaa
     1861 aaaaatccgg agaagaactg ctgagcaaaa attaccatct ggagaacgaa gttgcacggc
     1921 tgaagaaagg ctccggcagc ggaggaactc ctaaaaagaa acgcaaggtt aatgccacca
     1981 tggctagcgc ggtcggcgtg gactgtagaa cactgccaat gccggtccca agcccggata
     2041 aaagtggagg gtacagtcca cgctctagag cggacttcgg tccgcttttt actaggacct
     2101 gcaggcatgc aagcttgacg tcggttaccg atatccatat ggcgaccgca tcgatctcga
     2161 gccgaggact agtaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     2221 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     2281 catcaatgta tcttatcatg tcttacgtag ataagtagca tggcgggtta atcattaact
     2341 acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     2401 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     2461 agcgagcgcg cagagaggga cagatctgca gatctgctgg cgtaatagcg aagaggcccg
     2521 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt
     2581 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     2641 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     2701 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     2761 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     2821 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     2881 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     2941 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     3001 aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     3061 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3121 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3181 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3241 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     3301 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     3361 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     3421 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     3481 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     3541 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     3601 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3661 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     3721 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3781 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3841 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     3901 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3961 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     4021 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4081 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4141 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4201 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4261 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4321 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4381 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4441 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4501 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4561 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4621 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4681 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4741 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4801 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4861 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4921 gagcgcagcg agtcagtgag cgagcgcgca gagagggagt ggccaactcc atcactaggg
     4981 gttcctggag gggtggagtc gtgacgtgaa ttacgtcata gggttaggga ggtcctggat
     5041 cgatccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     5101 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     5161 ctgcaataaa caagttaaca acaacaattg cattcgtttt atgtttcagg ttcaggggga
     5221 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga
     5281 tctctagtca aggcactata catcaaatat tccttattaa cccctttaca aattaaaaag
     5341 ctaaaggtac acaatttttg agcatagtta ttaatagcag acactctatg cctgtgtgga
     5401 gtaagaaaaa acagtatgtt atgattataa ctgttatgcc tacttataaa ggttacagaa
     5461 tatttttcca taattttctt gtatagcagt gcagcttttt cctttgtggt gtaaatagca
     5521 aagcaagcaa gagttctatt actaaacaca gcatgactca aaaaacttag caattctgaa
     5581 ggaaagtcct tggggtcttc tacctttctc ttcttttttg gaggagtaga atgttgagag
     5641 tcagcagtag cctcatcatc actagatggc atttcttctg agcaaaacag gttttcctca
     5701 ttaaaggcat tccaccactg ctcccattca tcagttccat aggttggaat ctaaaataca
     5761 caaacaatta gaatcagtag tttaacacat tatacactta aaaattttat atttacctta
     5821 gagctttaaa tctctgtagg tagtttgtcc aattatgtca caccacagaa gtaaggttcc
     5881 ttcacaaaga tccggaccaa agcggccatc gtgcctcccc actcctgcag ttcgggggca
     5941 tggatgcgcg gatagccgct gctggtttcc tggatgccga cggatttgca ctgccggtag
     6001 aactccgcga ggtcgtccag cctcaggcag cagctgaacc aactcgcgag gggatcgagc
     6061 ccggggtggg cgaagaactc cagcatgaga tccccgcgct ggaggatcat ccagccggcg
     6121 tcccggaaaa cgattccgaa gcccaacctt tcatagaagg cggcggtgga atcgaaatct
     6181 cgtgatggca ggttgggcgt cgcttggtcg gtcatttcga accccagagt cccgctcaga
     6241 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt
     6301 aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag
     6361 ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag
     6421 aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg gtcacgacga
     6481 gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc
     6541 cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg
     6601 ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat
     6661 gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg
     6721 acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga
     6781 caacgtcgag cacagctgcg caaggaa
//
