LOCUS       sequence-458567-        7017 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express circular RNA translation reporter with 2xALFATag-10xSunTag 
            under H1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7017)
  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 7017)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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
FEATURES             Location/Qualifiers
     source          1..7017
                     /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             1323..1343
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1347..1356
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1547..1668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1717..1846
                     /label=AAV2 ITR
     rep_origin      complement(1940..2395)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2077..2098)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2289..2308
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2422..2526
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2527..2595,2596..3387)
                     /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(2745..2764)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3558..4146
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4047..4066
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4415..4549
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4974..4994)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          5124..5189
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(5609..6403)
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGYSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5720..5739)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6330..6349
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6760..7017,1..72))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6774..6909
                     /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 aggcggtgga ggcagtggta
     1321 cccccaagaa gaagcgcaag gtgaatgcca ccatggctag cgcggtcggc gtggactgta
     1381 gaacactgcc aatgccggtc ccaagcccgg ataaaagtgg agggtacagt ccacgctcta
     1441 gagcggactt cggtccgctt tttactagga cctgcaggca tgcaagcttg acgtcggtta
     1501 ccgatatcca tatggcgacc gcatcgatct cgagccgagg actagtaact tgtttattgc
     1561 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     1621 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtcttacg
     1681 tagataagta gcatggcggg ttaatcatta actacaagga acccctagtg atggagttgg
     1741 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     1801 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagagag ggacagatct
     1861 gcagatctgc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     1921 cagcctgaat ggcgaatgga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     1981 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     2041 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     2101 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     2161 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     2221 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     2281 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     2341 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     2401 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2461 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2521 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     2581 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2641 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2701 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2761 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     2821 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     2881 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     2941 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     3001 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     3061 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     3121 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     3181 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     3241 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     3301 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3361 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     3421 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     3481 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3541 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3601 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3661 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     3721 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3781 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3841 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3901 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3961 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4021 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4081 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4141 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4201 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4261 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgagcgc
     4321 gcagagaggg agtggccaac tccatcacta ggggttcctg gaggggtgga gtcgtgacgt
     4381 gaattacgtc atagggttag ggaggtcctg gatcgatcca gacatgataa gatacattga
     4441 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     4501 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     4561 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     4621 aaacctctac aaatgtggta tggctgatta tgatctctag tcaaggcact atacatcaaa
     4681 tattccttat taaccccttt acaaattaaa aagctaaagg tacacaattt ttgagcatag
     4741 ttattaatag cagacactct atgcctgtgt ggagtaagaa aaaacagtat gttatgatta
     4801 taactgttat gcctacttat aaaggttaca gaatattttt ccataatttt cttgtatagc
     4861 agtgcagctt tttcctttgt ggtgtaaata gcaaagcaag caagagttct attactaaac
     4921 acagcatgac tcaaaaaact tagcaattct gaaggaaagt ccttggggtc ttctaccttt
     4981 ctcttctttt ttggaggagt agaatgttga gagtcagcag tagcctcatc atcactagat
     5041 ggcatttctt ctgagcaaaa caggttttcc tcattaaagg cattccacca ctgctcccat
     5101 tcatcagttc cataggttgg aatctaaaat acacaaacaa ttagaatcag tagtttaaca
     5161 cattatacac ttaaaaattt tatatttacc ttagagcttt aaatctctgt aggtagtttg
     5221 tccaattatg tcacaccaca gaagtaaggt tccttcacaa agatccggac caaagcggcc
     5281 atcgtgcctc cccactcctg cagttcgggg gcatggatgc gcggatagcc gctgctggtt
     5341 tcctggatgc cgacggattt gcactgccgg tagaactccg cgaggtcgtc cagcctcagg
     5401 cagcagctga accaactcgc gaggggatcg agcccggggt gggcgaagaa ctccagcatg
     5461 agatccccgc gctggaggat catccagccg gcgtcccgga aaacgattcc gaagcccaac
     5521 ctttcataga aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg
     5581 tcggtcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg
     5641 cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt
     5701 cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg
     5761 ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat
     5821 tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct
     5881 tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct
     5941 gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt
     6001 ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga
     6061 tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc
     6121 ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa
     6181 cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac
     6241 cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg
     6301 cggcatcaga gtagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc
     6361 aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc
     6421 ctgtctcttg atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca
     6481 tccagtttac tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg
     6541 gttcgcttgc tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag
     6601 ctacctgctt tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat
     6661 tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc
     6721 agcccttgcg ccctgagtgc ttgcggcagc gtgaagcttt ttgcaaaagc ctaggcctcc
     6781 aaaaaagcct cctcactact tctggaatag ctcagaggcc gaggcggcct cggcctctgc
     6841 ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag
     6901 gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga gatgcatgct
     6961 ttgcatactt ctgcctgctg gggagcctgg ggactttcca cacctggttg ctgacta
//
