LOCUS       sequence-459315-        6579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express circular RNA translation reporter with 1xALFATag-5xSunTag 
            under H1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6579)
  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 6579)
  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-1xALFA-5xSunTag
FEATURES             Location/Qualifiers
     source          1..6579
                     /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             519..575
                     /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             591..647
                     /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             663..719
                     /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             735..791
                     /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             807..863
                     /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             885..905
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      909..918
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1109..1230
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1279..1408
                     /label=AAV2 ITR
     rep_origin      complement(1502..1957)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1639..1660)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1851..1870
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1984..2088
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2089..2157,2158..2949)
                     /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(2307..2326)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3120..3708
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3609..3628
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3977..4111
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4536..4556)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          4686..4751
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(5171..5965)
                     /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(5282..5301)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5892..5911
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6322..6579,1..72))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6336..6471
                     /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 ctaccggtga agagctacta tccaagaatt
      541 atcatcttga gaacgaggta gcgcgtttga agaagggttc cggctcagga gaggaactgc
      601 tctcgaagaa ctatcatctt gaaaatgagg tcgctcgatt aaaaaaggga tcgggcagtg
      661 gtgaggaact actttcaaag aattaccacc tcgaaaacga agtagctcga ttaaagaaag
      721 gttcagggtc gggtgaagaa ttactgagta aaaattatca tctggaaaat gaggtagcga
      781 gactaaaaaa ggggagtggt tctggcgagg aattgctatc gaaaaattat catcttgaga
      841 acgaagttgc taggctcaaa aagggctcag gctcaggcgg tacccccaag aagaagcgca
      901 aggtgaatgc caccatggct agcgcggtcg gcgtggactg tagaacactg ccaatgccgg
      961 tcccaagccc ggataaaagt ggagggtaca gtccacgctc tagagcggac ttcggtccgc
     1021 tttttactag gacctgcagg catgcaagct tgacgtcggt taccgatatc catatggcga
     1081 ccgcatcgat ctcgagccga ggactagtaa cttgtttatt gcagcttata atggttacaa
     1141 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     1201 tggtttgtcc aaactcatca atgtatctta tcatgtctta cgtagataag tagcatggcg
     1261 ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     1321 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     1381 cggcctcagt gagcgagcga gcgcgcagag agggacagat ctgcagatct gctggcgtaa
     1441 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     1501 gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     1561 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     1621 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     1681 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     1741 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     1801 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     1861 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     1921 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     1981 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2041 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2101 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2161 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2221 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2281 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     2341 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     2401 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     2461 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     2521 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     2581 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     2641 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     2701 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     2761 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     2821 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     2881 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     2941 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3001 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3061 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3121 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3181 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3241 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     3301 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3361 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3421 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3481 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3541 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3601 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3661 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3721 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3781 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3841 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagc gcgcagagag ggagtggcca
     3901 actccatcac taggggttcc tggaggggtg gagtcgtgac gtgaattacg tcatagggtt
     3961 agggaggtcc tggatcgatc cagacatgat aagatacatt gatgagtttg gacaaaccac
     4021 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4081 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     4141 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     4201 tatggctgat tatgatctct agtcaaggca ctatacatca aatattcctt attaacccct
     4261 ttacaaatta aaaagctaaa ggtacacaat ttttgagcat agttattaat agcagacact
     4321 ctatgcctgt gtggagtaag aaaaaacagt atgttatgat tataactgtt atgcctactt
     4381 ataaaggtta cagaatattt ttccataatt ttcttgtata gcagtgcagc tttttccttt
     4441 gtggtgtaaa tagcaaagca agcaagagtt ctattactaa acacagcatg actcaaaaaa
     4501 cttagcaatt ctgaaggaaa gtccttgggg tcttctacct ttctcttctt ttttggagga
     4561 gtagaatgtt gagagtcagc agtagcctca tcatcactag atggcatttc ttctgagcaa
     4621 aacaggtttt cctcattaaa ggcattccac cactgctccc attcatcagt tccataggtt
     4681 ggaatctaaa atacacaaac aattagaatc agtagtttaa cacattatac acttaaaaat
     4741 tttatattta ccttagagct ttaaatctct gtaggtagtt tgtccaatta tgtcacacca
     4801 cagaagtaag gttccttcac aaagatccgg accaaagcgg ccatcgtgcc tccccactcc
     4861 tgcagttcgg gggcatggat gcgcggatag ccgctgctgg tttcctggat gccgacggat
     4921 ttgcactgcc ggtagaactc cgcgaggtcg tccagcctca ggcagcagct gaaccaactc
     4981 gcgaggggat cgagcccggg gtgggcgaag aactccagca tgagatcccc gcgctggagg
     5041 atcatccagc cggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg
     5101 gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc
     5161 agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg
     5221 gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag
     5281 caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac
     5341 agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc
     5401 catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt
     5461 cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt
     5521 ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag
     5581 ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag
     5641 gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc
     5701 ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc
     5761 acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga
     5821 caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga
     5881 ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg
     5941 cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc
     6001 ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg
     6061 gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata
     6121 aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg
     6181 cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc
     6241 gtttctgcgg actggctttc tacgtgttcc gcttccttta gcagcccttg cgccctgagt
     6301 gcttgcggca gcgtgaagct ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta
     6361 cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa aaaaaattag
     6421 tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt
     6481 taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc
     6541 tggggagcct ggggactttc cacacctggt tgctgacta
//
