LOCUS       sequence-459302-        7095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express circular RNA translation reporter with 
            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 7095)
  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 7095)
  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-Tornado-2xALFA-10xSunTag-Xbp1(S255A)
FEATURES             Location/Qualifiers
     source          1..7095
                     /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"
     CDS             456..494
                     /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             516..554
                     /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             585..641
                     /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             657..713
                     /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             729..785
                     /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             801..857
                     /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             873..929
                     /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             945..1001
                     /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             1017..1073
                     /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             1089..1145
                     /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             1161..1217
                     /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             1233..1289
                     /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             1401..1421
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1425..1434
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1625..1746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1795..1924
                     /label=AAV2 ITR
     rep_origin      complement(2018..2473)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2155..2176)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2367..2386
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2500..2604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2605..2673,2674..3465)
                     /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(2823..2842)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3636..4224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4125..4144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4493..4627
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5052..5072)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          5202..5267
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(5687..6481)
                     /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(5798..5817)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6408..6427
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6838..7095,1..72))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6852..6987
                     /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 cgggccgcac tcgccggtcc caagcccgga taaaatggga gggggcggga aaccgcctaa
      421 ccataccgag tgcggccgca ggatctggat ctggaagcag gctggaggaa gagctgagga
      481 gacgcctgac tgagccaggt ggcggaggtt ctagtagcag actggaggag gagctgagaa
      541 gaagactgac cgagcccggt ggtggaggca gcaccggtgc ggaagaagaa cttttgagca
      601 agaattatca tcttgagaac gaagtggctc gtcttaagaa aggttctggc agtggagaag
      661 aactgctttc aaagaattac cacctggaaa atgaggtagc tagactgaaa aaggggagcg
      721 gaagtgggga ggagttgctg agcaaaaatt atcatttgga gaacgaagta gcacgactaa
      781 agaaagggtc cggatcgggt gaggagttac tctcgaaaaa ttatcatctc gaaaacgaag
      841 tggctcggct aaaaaagggc agtggttctg gagaagagct attatctaaa aactaccacc
      901 tcgaaaatga ggtggcacgc ttaaaaaagg gaagtggcag tggtgaagag ctactatcca
      961 agaattatca tcttgagaac gaggtagcgc gtttgaagaa gggttccggc tcaggagagg
     1021 aactgctctc gaagaactat catcttgaaa atgaggtcgc tcgattaaaa aagggatcgg
     1081 gcagtggtga ggaactactt tcaaagaatt accacctcga aaacgaagta gctcgattaa
     1141 agaaaggttc agggtcgggt gaagaattac tgagtaaaaa ttatcatctg gaaaatgagg
     1201 tagcgagact aaaaaagggg agtggttctg gcgaggaatt gctatcgaaa aattatcatc
     1261 ttgagaacga agttgctagg ctcaaaaagg gctcaggctc aggcggtaag gaccccgtgc
     1321 cctaccaacc tccattcctg tgtcaatggg gccgacatca gccagcctgg aaacccttga
     1381 tgaatggagg cagtggtacc cccaagaaga agcgcaaggt gaatgccacc atggctagcg
     1441 cggtcggcgt ggactgtaga acactgccaa tgccggtccc aagcccggat aaaagtggag
     1501 ggtacagtcc acgctctaga gcggacttcg gtccgctttt tactaggacc tgcaggcatg
     1561 caagcttgac gtcggttacc gatatccata tggcgaccgc atcgatctcg agccgaggac
     1621 tagtaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1681 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1741 atcttatcat gtcttacgta gataagtagc atggcgggtt aatcattaac tacaaggaac
     1801 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     1861 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     1921 gcagagaggg acagatctgc agatctgctg gcgtaatagc gaagaggccc gcaccgatcg
     1981 cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg
     2041 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     2101 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     2161 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     2221 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     2281 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     2341 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     2401 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     2461 ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2521 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     2581 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     2641 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     2701 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     2761 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     2821 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     2881 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2941 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     3001 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     3061 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     3121 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     3181 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     3241 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     3301 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     3361 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     3421 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     3481 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     3541 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     3601 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3661 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3721 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3781 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3841 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3901 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3961 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     4021 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4081 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4141 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4201 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4261 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     4321 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     4381 gagtcagtga gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgga
     4441 ggggtggagt cgtgacgtga attacgtcat agggttaggg aggtcctgga tcgatccaga
     4501 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     4561 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     4621 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     4681 ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg atctctagtc
     4741 aaggcactat acatcaaata ttccttatta acccctttac aaattaaaaa gctaaaggta
     4801 cacaattttt gagcatagtt attaatagca gacactctat gcctgtgtgg agtaagaaaa
     4861 aacagtatgt tatgattata actgttatgc ctacttataa aggttacaga atatttttcc
     4921 ataattttct tgtatagcag tgcagctttt tcctttgtgg tgtaaatagc aaagcaagca
     4981 agagttctat tactaaacac agcatgactc aaaaaactta gcaattctga aggaaagtcc
     5041 ttggggtctt ctacctttct cttctttttt ggaggagtag aatgttgaga gtcagcagta
     5101 gcctcatcat cactagatgg catttcttct gagcaaaaca ggttttcctc attaaaggca
     5161 ttccaccact gctcccattc atcagttcca taggttggaa tctaaaatac acaaacaatt
     5221 agaatcagta gtttaacaca ttatacactt aaaaatttta tatttacctt agagctttaa
     5281 atctctgtag gtagtttgtc caattatgtc acaccacaga agtaaggttc cttcacaaag
     5341 atccggacca aagcggccat cgtgcctccc cactcctgca gttcgggggc atggatgcgc
     5401 ggatagccgc tgctggtttc ctggatgccg acggatttgc actgccggta gaactccgcg
     5461 aggtcgtcca gcctcaggca gcagctgaac caactcgcga ggggatcgag cccggggtgg
     5521 gcgaagaact ccagcatgag atccccgcgc tggaggatca tccagccggc gtcccggaaa
     5581 acgattccga agcccaacct ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc
     5641 aggttgggcg tcgcttggtc ggtcatttcg aaccccagag tcccgctcag aagaactcgt
     5701 caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga
     5761 ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta
     5821 tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc
     5881 cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc
     5941 cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct
     6001 cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga
     6061 tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc
     6121 gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat gacaggagat
     6181 cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga
     6241 gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct
     6301 gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg
     6361 ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc
     6421 cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca
     6481 tgcgaaacga tcctcatcct gtctcttgat cagatcttga tcccctgcgc catcagatcc
     6541 ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg
     6601 ccccagctgg caattccggt tcgcttgctg tccataaaac cgcccagtct agctatcgcc
     6661 atgtaagccc actgcaagct acctgctttc tctttgcgct tgcgttttcc cttgtccaga
     6721 tagcccagta gctgacattc atccggggtc agcaccgttt ctgcggactg gctttctacg
     6781 tgttccgctt cctttagcag cccttgcgcc ctgagtgctt gcggcagcgt gaagcttttt
     6841 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga
     6901 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
     6961 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
     7021 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     7081 cctggttgct gacta
//
