LOCUS       sequence-468947-        6741 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express circular RNA translation reporter with 1xALFATag-5xSunTag 
            under CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6741)
  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 6741)
  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-CMVp-2xTetO-Tornado-1xALFATag-5xSunTag
FEATURES             Location/Qualifiers
     source          1..6741
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     150..169
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     enhancer        607..986
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        987..1190
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    1192..1210
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1213..1231
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1241..1265
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1455..1493
                     /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             1518..1574
                     /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             1590..1646
                     /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             1662..1718
                     /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             1734..1790
                     /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             1806..1862
                     /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             1884..1904
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1908..1917
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2108..2229
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2278..2407
                     /label=AAV2 ITR
     rep_origin      complement(2501..2956)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2638..2659)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2850..2869
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2983..3087
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3088..3156,3157..3948)
                     /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(3306..3325)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4119..4707
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4608..4627
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4976..5110
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5535..5555)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          5685..5750
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(join(6170..6741,1..223))
                     /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(6281..6300)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac
       61 cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg
      121 cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc
      181 aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc
      241 ctgtctcttg atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca
      301 tccagtttac tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg
      361 gttcgcttgc tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag
      421 ctacctgctt tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat
      481 tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc
      541 agcccttgcg ccctgagtgc ttgcggcagc gtgaagcttt ttgcaaaagc taggcctggg
      601 atccttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      661 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      721 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      781 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      841 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      901 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      961 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     1021 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     1081 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     1141 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca
     1201 gtgatagaga tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc
     1261 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
     1321 ccagcctccg gactctagcg tttaaacgcg tactgtcgac gggccgcact cgccggtccc
     1381 aagcccggat aaaatgggag ggggcgggaa accgcctaac cataccgagt gcggccgcag
     1441 gatctggatc tggaagcagg ctggaggaag agctgaggag acgcctgact gagccaggtg
     1501 gcggaggttc taccggtgaa gagctactat ccaagaatta tcatcttgag aacgaggtag
     1561 cgcgtttgaa gaagggttcc ggctcaggag aggaactgct ctcgaagaac tatcatcttg
     1621 aaaatgaggt cgctcgatta aaaaagggat cgggcagtgg tgaggaacta ctttcaaaga
     1681 attaccacct cgaaaacgaa gtagctcgat taaagaaagg ttcagggtcg ggtgaagaat
     1741 tactgagtaa aaattatcat ctggaaaatg aggtagcgag actaaaaaag gggagtggtt
     1801 ctggcgagga attgctatcg aaaaattatc atcttgagaa cgaagttgct aggctcaaaa
     1861 agggctcagg ctcaggcggt acccccaaga agaagcgcaa ggtgaatgcc accatggcta
     1921 gcgcggtcgg cgtggactgt agaacactgc caatgccggt cccaagcccg gataaaagtg
     1981 gagggtacag tccacgctct agagcggact tcggtccgct ttttactagg acctgcaggc
     2041 atgcaagctt gacgtcggtt accgatatcc atatggcgac cgcatcgatc tcgagccgag
     2101 gactagtaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2161 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2221 tgtatcttat catgtcttac gtagataagt agcatggcgg gttaatcatt aactacaagg
     2281 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     2341 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     2401 cgcgcagaga gggacagatc tgcagatctg ctggcgtaat agcgaagagg cccgcaccga
     2461 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt
     2521 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2581 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2641 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2701 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2761 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2821 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2881 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     2941 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3001 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3061 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     3121 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3181 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     3241 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     3301 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     3361 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     3421 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     3481 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     3541 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     3601 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     3661 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     3721 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     3781 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     3841 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     3901 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     3961 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4021 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4081 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4141 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4201 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4261 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4321 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4381 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4441 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4501 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4561 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4621 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4681 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4741 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4801 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4861 agcgagtcag tgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
     4921 ggaggggtgg agtcgtgacg tgaattacgt catagggtta gggaggtcct ggatcgatcc
     4981 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     5041 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     5101 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     5161 ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt atgatctcta
     5221 gtcaaggcac tatacatcaa atattcctta ttaacccctt tacaaattaa aaagctaaag
     5281 gtacacaatt tttgagcata gttattaata gcagacactc tatgcctgtg tggagtaaga
     5341 aaaaacagta tgttatgatt ataactgtta tgcctactta taaaggttac agaatatttt
     5401 tccataattt tcttgtatag cagtgcagct ttttcctttg tggtgtaaat agcaaagcaa
     5461 gcaagagttc tattactaaa cacagcatga ctcaaaaaac ttagcaattc tgaaggaaag
     5521 tccttggggt cttctacctt tctcttcttt tttggaggag tagaatgttg agagtcagca
     5581 gtagcctcat catcactaga tggcatttct tctgagcaaa acaggttttc ctcattaaag
     5641 gcattccacc actgctccca ttcatcagtt ccataggttg gaatctaaaa tacacaaaca
     5701 attagaatca gtagtttaac acattataca cttaaaaatt ttatatttac cttagagctt
     5761 taaatctctg taggtagttt gtccaattat gtcacaccac agaagtaagg ttccttcaca
     5821 aagatccgga ccaaagcggc catcgtgcct ccccactcct gcagttcggg ggcatggatg
     5881 cgcggatagc cgctgctggt ttcctggatg ccgacggatt tgcactgccg gtagaactcc
     5941 gcgaggtcgt ccagcctcag gcagcagctg aaccaactcg cgaggggatc gagcccgggg
     6001 tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg
     6061 aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat
     6121 ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact
     6181 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     6241 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     6301 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc
     6361 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct
     6421 cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     6481 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
     6541 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
     6601 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
     6661 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
     6721 cgagcacagc tgcgcaagga a
//
