LOCUS       sequence-459551-        7259 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express high ribosome loading circular RNA translation reporter 
            2xALFATag-10xSunTag-2xXbp1(S255A) pause site under H1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7259)
  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 7259)
  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-2x
            Xbp1(S255A)
FEATURES             Location/Qualifiers
     source          1..7259
                     /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             893..949
                     /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             965..1021
                     /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             1037..1093
                     /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             1109..1165
                     /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             1181..1237
                     /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             1253..1309
                     /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             1325..1381
                     /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             1397..1453
                     /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             1565..1585
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1589..1598
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1789..1910
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1959..2088
                     /label=AAV2 ITR
     rep_origin      complement(2182..2637)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2319..2340)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2531..2550
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2664..2768
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2769..2837,2838..3629)
                     /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(2987..3006)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3800..4388
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4289..4308
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4657..4791
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5216..5236)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          5366..5431
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(5851..6645)
                     /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(5962..5981)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6572..6591
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(7002..7259,1..72))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7016..7151
                     /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 gagcaaggac cccgtgccct accaacctcc
      841 attcctgtgt caatggggcc gacatcagcc agcctggaaa cccttgatga atgaggagtt
      901 gctgagcaaa aattatcatt tggagaacga agtagcacga ctaaagaaag ggtccggatc
      961 gggtgaggag ttactctcga aaaattatca tctcgaaaac gaagtggctc ggctaaaaaa
     1021 gggcagtggt tctggagaag agctattatc taaaaactac cacctcgaaa atgaggtggc
     1081 acgcttaaaa aagggaagtg gcagtggtga agagctacta tccaagaatt atcatcttga
     1141 gaacgaggta gcgcgtttga agaagggttc cggctcagga gaggaactgc tctcgaagaa
     1201 ctatcatctt gaaaatgagg tcgctcgatt aaaaaaggga tcgggcagtg gtgaggaact
     1261 actttcaaag aattaccacc tcgaaaacga agtagctcga ttaaagaaag gttcagggtc
     1321 gggtgaagaa ttactgagta aaaattatca tctggaaaat gaggtagcga gactaaaaaa
     1381 ggggagtggt tctggcgagg aattgctatc gaaaaattat catcttgaga acgaagttgc
     1441 taggctcaaa aagggctcag gctcaggcgg taaggacccc gtgccctacc aacctccatt
     1501 cctgtgtcaa tggggccgac atcagccagc ctggaaaccc ttgatgaatg gaggcagtgg
     1561 tacccccaag aagaagcgca aggtgaatgc caccatggct agcgcggtcg gcgtggactg
     1621 tagaacactg ccaatgccgg tcccaagccc ggataaaagt ggagggtaca gtccacgctc
     1681 tagagcggac ttcggtccgc tttttactag gacctgcagg catgcaagct tgacgtcggt
     1741 taccgatatc catatggcga ccgcatcgat ctcgagccga ggactagtaa cttgtttatt
     1801 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     1861 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctta
     1921 cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt
     1981 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     2041 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagag agggacagat
     2101 ctgcagatct gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     2161 cgcagcctga atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     2221 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     2281 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     2341 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     2401 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     2461 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     2521 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     2581 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag
     2641 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2701 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2761 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2821 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2881 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2941 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3001 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3061 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3121 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3181 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3241 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3301 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3361 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3421 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3481 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3541 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3601 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3661 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3721 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3781 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3841 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3901 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     3961 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4021 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4081 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4141 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4201 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4261 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4321 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4381 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4441 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4501 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagc
     4561 gcgcagagag ggagtggcca actccatcac taggggttcc tggaggggtg gagtcgtgac
     4621 gtgaattacg tcatagggtt agggaggtcc tggatcgatc cagacatgat aagatacatt
     4681 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     4741 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     4801 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag
     4861 taaaacctct acaaatgtgg tatggctgat tatgatctct agtcaaggca ctatacatca
     4921 aatattcctt attaacccct ttacaaatta aaaagctaaa ggtacacaat ttttgagcat
     4981 agttattaat agcagacact ctatgcctgt gtggagtaag aaaaaacagt atgttatgat
     5041 tataactgtt atgcctactt ataaaggtta cagaatattt ttccataatt ttcttgtata
     5101 gcagtgcagc tttttccttt gtggtgtaaa tagcaaagca agcaagagtt ctattactaa
     5161 acacagcatg actcaaaaaa cttagcaatt ctgaaggaaa gtccttgggg tcttctacct
     5221 ttctcttctt ttttggagga gtagaatgtt gagagtcagc agtagcctca tcatcactag
     5281 atggcatttc ttctgagcaa aacaggtttt cctcattaaa ggcattccac cactgctccc
     5341 attcatcagt tccataggtt ggaatctaaa atacacaaac aattagaatc agtagtttaa
     5401 cacattatac acttaaaaat tttatattta ccttagagct ttaaatctct gtaggtagtt
     5461 tgtccaatta tgtcacacca cagaagtaag gttccttcac aaagatccgg accaaagcgg
     5521 ccatcgtgcc tccccactcc tgcagttcgg gggcatggat gcgcggatag ccgctgctgg
     5581 tttcctggat gccgacggat ttgcactgcc ggtagaactc cgcgaggtcg tccagcctca
     5641 ggcagcagct gaaccaactc gcgaggggat cgagcccggg gtgggcgaag aactccagca
     5701 tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca
     5761 acctttcata gaaggcggcg gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt
     5821 ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa
     5881 ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca
     5941 ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc
     6001 cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat
     6061 attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc
     6121 cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc
     6181 ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg
     6241 gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat
     6301 gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc
     6361 gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg
     6421 aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc
     6481 accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac
     6541 ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac
     6601 ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca
     6661 tcctgtctct tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc
     6721 catccagttt actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc
     6781 cggttcgctt gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca
     6841 agctacctgc tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac
     6901 attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtgttcc gcttccttta
     6961 gcagcccttg cgccctgagt gcttgcggca gcgtgaagct ttttgcaaaa gcctaggcct
     7021 ccaaaaaagc ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct
     7081 gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt
     7141 aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg
     7201 ctttgcatac ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgacta
//
