LOCUS       sequence-459553-        7092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express circular RNA translation reporter with 
            2xALFATag-10xSunTag-Pseudoknot under H1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7092)
  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 7092)
  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-Pseudoknot
FEATURES             Location/Qualifiers
     source          1..7092
                     /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             1398..1418
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1422..1431
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1622..1743
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1792..1921
                     /label=AAV2 ITR
     rep_origin      complement(2015..2470)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2152..2173)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2364..2383
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2497..2601
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2602..2670,2671..3462)
                     /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(2820..2839)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3633..4221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4122..4141
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4490..4624
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5049..5069)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          5199..5264
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(5684..6478)
                     /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(5795..5814)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6405..6424
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6835..7092,1..72))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6849..6984
                     /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 gggttccagc 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 aggcggtgga ggcagtggta
     1321 cagtcaggcc ggaaaattcc cgccaccgga agttgagtag acggtgctgc ctgcgactca
     1381 accccaggag gactgggccc aagaagaagc gcaaggtgaa tgccaccatg gctagcgcgg
     1441 tcggcgtgga ctgtagaaca ctgccaatgc cggtcccaag cccggataaa agtggagggt
     1501 acagtccacg ctctagagcg gacttcggtc cgctttttac taggacctgc aggcatgcaa
     1561 gcttgacgtc ggttaccgat atccatatgg cgaccgcatc gatctcgagc cgaggactag
     1621 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     1681 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     1741 ttatcatgtc ttacgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc
     1801 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     1861 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     1921 gagagggaca gatctgcaga tctgctggcg taatagcgaa gaggcccgca ccgatcgccc
     1981 ttcccaacag ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta
     2041 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     2101 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     2161 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     2221 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     2281 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     2341 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     2401 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     2461 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2521 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2581 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     2641 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     2701 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     2761 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     2821 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     2881 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     2941 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     3001 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3061 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3121 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     3181 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3241 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3301 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     3361 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3421 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3481 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     3541 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3601 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3661 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3721 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     3781 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3841 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3901 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3961 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4021 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4081 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4141 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4201 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4261 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4321 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4381 tcagtgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctggaggg
     4441 gtggagtcgt gacgtgaatt acgtcatagg gttagggagg tcctggatcg atccagacat
     4501 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     4561 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     4621 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     4681 tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc tctagtcaag
     4741 gcactataca tcaaatattc cttattaacc cctttacaaa ttaaaaagct aaaggtacac
     4801 aatttttgag catagttatt aatagcagac actctatgcc tgtgtggagt aagaaaaaac
     4861 agtatgttat gattataact gttatgccta cttataaagg ttacagaata tttttccata
     4921 attttcttgt atagcagtgc agctttttcc tttgtggtgt aaatagcaaa gcaagcaaga
     4981 gttctattac taaacacagc atgactcaaa aaacttagca attctgaagg aaagtccttg
     5041 gggtcttcta cctttctctt cttttttgga ggagtagaat gttgagagtc agcagtagcc
     5101 tcatcatcac tagatggcat ttcttctgag caaaacaggt tttcctcatt aaaggcattc
     5161 caccactgct cccattcatc agttccatag gttggaatct aaaatacaca aacaattaga
     5221 atcagtagtt taacacatta tacacttaaa aattttatat ttaccttaga gctttaaatc
     5281 tctgtaggta gtttgtccaa ttatgtcaca ccacagaagt aaggttcctt cacaaagatc
     5341 cggaccaaag cggccatcgt gcctccccac tcctgcagtt cgggggcatg gatgcgcgga
     5401 tagccgctgc tggtttcctg gatgccgacg gatttgcact gccggtagaa ctccgcgagg
     5461 tcgtccagcc tcaggcagca gctgaaccaa ctcgcgaggg gatcgagccc ggggtgggcg
     5521 aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg
     5581 attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tgatggcagg
     5641 ttgggcgtcg cttggtcggt catttcgaac cccagagtcc cgctcagaag aactcgtcaa
     5701 gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga
     5761 agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt
     5821 cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat
     5881 tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt
     5941 cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt
     6001 cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc
     6061 gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca
     6121 ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct
     6181 gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca
     6241 cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca
     6301 gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg
     6361 acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga
     6421 atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc
     6481 gaaacgatcc tcatcctgtc tcttgatcag atcttgatcc cctgcgccat cagatccttg
     6541 gcggcaagaa agccatccag tttactttgc agggcttccc aaccttacca gagggcgccc
     6601 cagctggcaa ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg
     6661 taagcccact gcaagctacc tgctttctct ttgcgcttgc gttttccctt gtccagatag
     6721 cccagtagct gacattcatc cggggtcagc accgtttctg cggactggct ttctacgtgt
     6781 tccgcttcct ttagcagccc ttgcgccctg agtgcttgcg gcagcgtgaa gctttttgca
     6841 aaagcctagg cctccaaaaa agcctcctca ctacttctgg aatagctcag aggccgaggc
     6901 ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga
     6961 actgggcgga gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta
     7021 attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct
     7081 ggttgctgac ta
//
