LOCUS       sequence-459554-        7166 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express high ribosome loading circular RNA translation reporter 
            2xALFATag-10xSunTag-Xbp1(S255A) pause site under H1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7166)
  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 7166)
  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-Xb
            p1(S255A)
FEATURES             Location/Qualifiers
     source          1..7166
                     /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             1472..1492
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1496..1505
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1696..1817
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1866..1995
                     /label=AAV2 ITR
     rep_origin      complement(2089..2544)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2226..2247)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2438..2457
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2571..2675
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2676..2744,2745..3536)
                     /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(2894..2913)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3707..4295
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4196..4215
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4564..4698
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5123..5143)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          5273..5338
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(5758..6552)
                     /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(5869..5888)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6479..6498
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6909..7166,1..72))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6923..7058
                     /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 caggcggtaa
     1381 ggaccccgtg ccctaccaac ctccattcct gtgtcaatgg ggccgacatc agccagcctg
     1441 gaaacccttg atgaatggag gcagtggtac ccccaagaag aagcgcaagg tgaatgccac
     1501 catggctagc gcggtcggcg tggactgtag aacactgcca atgccggtcc caagcccgga
     1561 taaaagtgga gggtacagtc cacgctctag agcggacttc ggtccgcttt ttactaggac
     1621 ctgcaggcat gcaagcttga cgtcggttac cgatatccat atggcgaccg catcgatctc
     1681 gagccgagga ctagtaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     1741 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     1801 ctcatcaatg tatcttatca tgtcttacgt agataagtag catggcgggt taatcattaa
     1861 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     1921 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     1981 cgagcgagcg cgcagagagg gacagatctg cagatctgct ggcgtaatag cgaagaggcc
     2041 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggaa attgtaagcg
     2101 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     2161 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     2221 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     2281 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     2341 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     2401 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     2461 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     2521 ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     2581 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     2641 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     2701 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     2761 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     2821 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     2881 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     2941 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3001 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3061 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3121 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3181 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     3241 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3301 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3361 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     3421 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     3481 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3541 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3601 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3661 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3721 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3781 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3841 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3901 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3961 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4021 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4081 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4141 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4201 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4261 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4321 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4381 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4441 ccgagcgcag cgagtcagtg agcgagcgcg cagagaggga gtggccaact ccatcactag
     4501 gggttcctgg aggggtggag tcgtgacgtg aattacgtca tagggttagg gaggtcctgg
     4561 atcgatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
     4621 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     4681 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     4741 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat
     4801 gatctctagt caaggcacta tacatcaaat attccttatt aaccccttta caaattaaaa
     4861 agctaaaggt acacaatttt tgagcatagt tattaatagc agacactcta tgcctgtgtg
     4921 gagtaagaaa aaacagtatg ttatgattat aactgttatg cctacttata aaggttacag
     4981 aatatttttc cataattttc ttgtatagca gtgcagcttt ttcctttgtg gtgtaaatag
     5041 caaagcaagc aagagttcta ttactaaaca cagcatgact caaaaaactt agcaattctg
     5101 aaggaaagtc cttggggtct tctacctttc tcttcttttt tggaggagta gaatgttgag
     5161 agtcagcagt agcctcatca tcactagatg gcatttcttc tgagcaaaac aggttttcct
     5221 cattaaaggc attccaccac tgctcccatt catcagttcc ataggttgga atctaaaata
     5281 cacaaacaat tagaatcagt agtttaacac attatacact taaaaatttt atatttacct
     5341 tagagcttta aatctctgta ggtagtttgt ccaattatgt cacaccacag aagtaaggtt
     5401 ccttcacaaa gatccggacc aaagcggcca tcgtgcctcc ccactcctgc agttcggggg
     5461 catggatgcg cggatagccg ctgctggttt cctggatgcc gacggatttg cactgccggt
     5521 agaactccgc gaggtcgtcc agcctcaggc agcagctgaa ccaactcgcg aggggatcga
     5581 gcccggggtg ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg
     5641 cgtcccggaa aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat
     5701 ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc gaaccccaga gtcccgctca
     5761 gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc
     5821 gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt
     5881 agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc
     5941 agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac
     6001 gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag
     6061 cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg
     6121 tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt
     6181 atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga
     6241 tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt
     6301 gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc
     6361 tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg
     6421 gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc
     6481 ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc
     6541 ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga tcagatcttg atcccctgcg
     6601 ccatcagatc cttggcggca agaaagccat ccagtttact ttgcagggct tcccaacctt
     6661 accagagggc gccccagctg gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc
     6721 tagctatcgc catgtaagcc cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc
     6781 ccttgtccag atagcccagt agctgacatt catccggggt cagcaccgtt tctgcggact
     6841 ggctttctac gtgttccgct tcctttagca gcccttgcgc cctgagtgct tgcggcagcg
     6901 tgaagctttt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc
     6961 tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc
     7021 ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact
     7081 atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     7141 gactttccac acctggttgc tgacta
//
