LOCUS       sequence-459529-        7088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express high ribosome loading circular RNA translation reporter with
            2xALFATag-10xSunTag under H1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7088)
  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 7088)
  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-H1promoter-2xTetO-High_ribosome_load-Tornado-2xALFA-10xSunTag
FEATURES             Location/Qualifiers
     source          1..7088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     57..77
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        229..323
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     240..259
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    277..295
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    305..323
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      401..410
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             527..565
                     /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             587..625
                     /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             656..712
                     /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             728..784
                     /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             800..856
                     /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             872..928
                     /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             944..1000
                     /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             1016..1072
                     /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             1088..1144
                     /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             1160..1216
                     /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             1232..1288
                     /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             1304..1360
                     /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             1394..1414
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1418..1427
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1618..1739
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1788..1917
                     /label=AAV2 ITR
     rep_origin      complement(2011..2466)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2148..2169)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2360..2379
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2493..2597
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2598..2666,2667..3458)
                     /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(2816..2835)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3629..4217
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4118..4137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4486..4620
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5045..5065)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          5195..5260
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(5680..6474)
                     /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(5791..5810)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6401..6420
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6831..7088,1..72))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6845..6980
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact ttccacaccc
       61 taactgacac acattccaca gccggatcca aggtcgggca ggaagaacgc tgacgtcatc
      121 aacccgctcc aaggaatcgc gggcccagtg tcactaggcg ggaacaccca gcgcgcgtgc
      181 gccctggcag gaagatggct gtgagggaca ggggagtggc gccctgcaat atttgcatgt
      241 cgctatgtgt tctgggaaat caccataaac gtgaaatccc tatcagtgat agagacttat
      301 aagttcccta tcagtgatag agatcccggt gctcgcttcg gcagcacata tactagtcga
      361 cgactctagc gtttaaaggt tcagtcacaa ttcacagata gccaccatgg tacgcgtact
      421 gtcgacgtcg acgggccgca ctcgccggtc ccaagcccgg ataaaatggg agggggcggg
      481 aaaccgccta accataccga gtgcggccgc aggatctgga tctggaagca ggctggagga
      541 agagctgagg agacgcctga ctgagccagg tggcggaggt tctagtagca gactggagga
      601 ggagctgaga agaagactga ccgagcccgg tggtggaggc agcaccggtg cggaagaaga
      661 acttttgagc aagaattatc atcttgagaa cgaagtggct cgtcttaaga aaggttctgg
      721 cagtggagaa gaactgcttt caaagaatta ccacctggaa aatgaggtag ctagactgaa
      781 aaaggggagc ggaagtgggg aggagttgct gagcaaaaat tatcatttgg agaacgaagt
      841 agcacgacta aagaaagggt ccggatcggg tgaggagtta ctctcgaaaa attatcatct
      901 cgaaaacgaa gtggctcggc taaaaaaggg cagtggttct ggagaagagc tattatctaa
      961 aaactaccac ctcgaaaatg aggtggcacg cttaaaaaag ggaagtggca gtggtgaaga
     1021 gctactatcc aagaattatc atcttgagaa cgaggtagcg cgtttgaaga agggttccgg
     1081 ctcaggagag gaactgctct cgaagaacta tcatcttgaa aatgaggtcg ctcgattaaa
     1141 aaagggatcg ggcagtggtg aggaactact ttcaaagaat taccacctcg aaaacgaagt
     1201 agctcgatta aagaaaggtt cagggtcggg tgaagaatta ctgagtaaaa attatcatct
     1261 ggaaaatgag gtagcgagac taaaaaaggg gagtggttct ggcgaggaat tgctatcgaa
     1321 aaattatcat cttgagaacg aagttgctag gctcaaaaag ggctcaggct caggcggtgg
     1381 aggcagtggt acccccaaga agaagcgcaa ggtgaatgcc accatggcta gcgcggtcgg
     1441 cgtggactgt agaacactgc caatgccggt cccaagcccg gataaaagtg gagggtacag
     1501 tccacgctct agagcggact tcggtccgct ttttactagg acctgcaggc atgcaagctt
     1561 gacgtcggtt accgatatcc atatggcgac cgcatcgatc tcgagccgag gactagtaac
     1621 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     1681 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     1741 catgtcttac gtagataagt agcatggcgg gttaatcatt aactacaagg aacccctagt
     1801 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     1861 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagaga
     1921 gggacagatc tgcagatctg ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     1981 caacagttgc gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt ttgttaaaat
     2041 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     2101 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     2161 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     2221 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     2281 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     2341 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     2401 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     2461 gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2521 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2581 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     2641 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2701 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     2761 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     2821 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     2881 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     2941 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3001 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3061 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3121 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3181 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3241 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3301 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3361 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3421 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3481 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3541 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3601 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3661 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3721 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     3781 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3841 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3901 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3961 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4021 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4081 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4141 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4201 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4261 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4321 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4381 tgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct ggaggggtgg
     4441 agtcgtgacg tgaattacgt catagggtta gggaggtcct ggatcgatcc agacatgata
     4501 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     4561 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     4621 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
     4681 taaagcaagt aaaacctcta caaatgtggt atggctgatt atgatctcta gtcaaggcac
     4741 tatacatcaa atattcctta ttaacccctt tacaaattaa aaagctaaag gtacacaatt
     4801 tttgagcata gttattaata gcagacactc tatgcctgtg tggagtaaga aaaaacagta
     4861 tgttatgatt ataactgtta tgcctactta taaaggttac agaatatttt tccataattt
     4921 tcttgtatag cagtgcagct ttttcctttg tggtgtaaat agcaaagcaa gcaagagttc
     4981 tattactaaa cacagcatga ctcaaaaaac ttagcaattc tgaaggaaag tccttggggt
     5041 cttctacctt tctcttcttt tttggaggag tagaatgttg agagtcagca gtagcctcat
     5101 catcactaga tggcatttct tctgagcaaa acaggttttc ctcattaaag gcattccacc
     5161 actgctccca ttcatcagtt ccataggttg gaatctaaaa tacacaaaca attagaatca
     5221 gtagtttaac acattataca cttaaaaatt ttatatttac cttagagctt taaatctctg
     5281 taggtagttt gtccaattat gtcacaccac agaagtaagg ttccttcaca aagatccgga
     5341 ccaaagcggc catcgtgcct ccccactcct gcagttcggg ggcatggatg cgcggatagc
     5401 cgctgctggt ttcctggatg ccgacggatt tgcactgccg gtagaactcc gcgaggtcgt
     5461 ccagcctcag gcagcagctg aaccaactcg cgaggggatc gagcccgggg tgggcgaaga
     5521 actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg aaaacgattc
     5581 cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg
     5641 gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact cgtcaagaag
     5701 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     5761 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     5821 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     5881 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     5941 catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     6001 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     6061 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     6121 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     6181 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     6241 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cctgcagttc
     6301 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     6361 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     6421 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     6481 cgatcctcat cctgtctctt gatcagatct tgatcccctg cgccatcaga tccttggcgg
     6541 caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc
     6601 tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc gccatgtaag
     6661 cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc agatagccca
     6721 gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct acgtgttccg
     6781 cttcctttag cagcccttgc gccctgagtg cttgcggcag cgtgaagctt tttgcaaaag
     6841 cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc cgaggcggcc
     6901 tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg
     6961 ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg
     7021 agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt
     7081 gctgacta
//
