LOCUS       sequence-492404-        7347 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  Express 42xRCGG/CGA/AGG mRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7347)
  AUTHORS   Zhu X, Cruz VE, Zhang H, Erzberger JP, Mendell JT
  TITLE     Specific tRNAs promote mRNA decay by recruiting the CCR4-NOT complex
            to translating ribosomes.
  JOURNAL   Science. 2024 Nov 22;386(6724):eadq8587. doi: 
            10.1126/science.adq8587. Epub 2024 Nov 22.
  PUBMED    39571015
REFERENCE   2  (bases 1 to 7347)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 10, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science."; 
            date: "2024-11-22"; volume: "386(6724):eadq8587. doi"; pages: " 
            10.1126/science.adq8587. Epub 2024 Nov 22"
COMMENT     Sequence Label: Xlone-tTA-42xRCGG/CGA/AGG
FEATURES             Location/Qualifiers
     source          1..7347
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..376
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      416..425
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2048..2182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2458..2492
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2503..2520)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2503..2519)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2512..2534)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2728..2747)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2847..2869
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2907..2925)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2993..3097
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3098..3166,3167..3958)
                     /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(3316..3335)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4129..4717
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4618..4637
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4871..4888
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5005..5026
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5041..5046,5047..5064,5065..5071)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5079..5095
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5084..5106
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5103..5119
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5103..5119
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5145..5207
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5383..5464
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5581..5979)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(5980..6036)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(join(6046..6429,6430..7050))
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     6958..6977
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             complement(7051..7074)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7074..7083
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7103..7314)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag ctttgcttat gtaaaccagg gcgcctataa aagagtgctg
      301 attttttgag taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac
      361 ttcctaccct cgtaaaggta ccgagctcaa gcttcgaatt ctgcagtcga cggccgccac
      421 catggtgctt cgggattcaa ggggtttacg ggatttacgg ggagttcggg aactacgaga
      481 tcctcgggac ttgcgaggcc ttcgggagct acgatctagt cgggatggac gaggcctccg
      541 ggaaacaagg tcgctccggg atctgcgggg agttcgagag agtcgggatg ttaggagtct
      601 ccgggaagtt cggggtgtta gggattatcg agagttacgg agtctgcggg gatataggga
      661 tagtagggaa tctcggagtg caaggggact acgggacctc aggtcaccac gggaatatcg
      721 aggattgcgg gatgttaggt ctctacggga gcttcgaggc ccacgagatt tgcggtcgtt
      781 aaggggagtc agggagctac gagaataatc tagattctgc acgtggatcc tgagaacttc
      841 agggtgagtc tatgggaccc ttgatgtttt ctttcccctt cttttctatg gttaagttca
      901 tgtcatagga aggggataag taacagggta cagtttagaa tgggaaacag acgaatgatt
      961 gcatcagtgt ggaagtctca ggatcgtttt agtttctttt atttgctgtt cataacaatt
     1021 gttttctttt gtttaattct tgctttcttt ttttttcttc tccgcaattt ttactattat
     1081 acttaatgcc ttaacattgt gtataacaaa aggaaatatc tctgagatac attaagtaac
     1141 ttaaaaaaaa actttacaca gtctgcctag tacattacta tttggaatat atgtgtgctt
     1201 atttgcatat tcataatctc cctactttat tttcttttat ttttaattga tacataatca
     1261 ttatacatat ttatgggtta aagtgtaatg ttttaatatg tgtacacata ttgaccaaat
     1321 cagggtaatt ttgcatttgt aattttaaaa aatgctttct tcttttaata tacttttttg
     1381 tttatcttat ttctaatact ttccctaatc tctttctttc agggcaataa tgatacaatg
     1441 tatcatgcct ctttgcacca ttctaaagaa taacagtgat aatttctggg ttaaggcaat
     1501 agcaatattt ctgcatataa atatttctgc atataaattg taactgatgt aagaggtttc
     1561 atattgctaa tagcagctac aatccagcta ccattctgct tttattttat ggttgggata
     1621 aggctggatt attctgagtc caagctaggc ccttttgcta atcatgttca tacctcttat
     1681 cttcctccca cagctcctgg gcaacgtgct ggtctgtgtg ctggcccatc actttggcaa
     1741 agaattcacc ccaccagtgc aggctgccta tcagaaagtg gtggctggtg tggctaatgc
     1801 cctggcccac aagtatcact aagctcgctt tcttgctgtc caatttctac aattgttgtt
     1861 gttaaacact agtagaccac ctcccctgcg agctaagctg gacagccaat gacgggtaag
     1921 agagtgacat ttttcactaa cctaagacag gagggccgtc agagctactg cctaatccaa
     1981 agacgggtaa aagtgataaa aatgtatcac tccaacctaa gacaggcgca gcttccgagg
     2041 gatttgagat ccagacatga taagatacat tgatgagttt ggacaaacca aaactagaat
     2101 gcagtgaaaa aaatgcctta tttgtgaaat ttgtgatgct attgccttat ttgtaaccat
     2161 tataagctgc aataaacaag tttgatatct ataacaagaa aatatatata taataagtta
     2221 tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat cttaaaagtc
     2281 acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc aaaatcagtg
     2341 acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact gagatgtcct
     2401 aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc aagaatgcat
     2461 gcgtcaattt tacgcagact atctttctag ggttaagaat tcactggccg tcgttttaca
     2521 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     2581 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     2641 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2701 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     2761 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     2821 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     2881 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2941 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     3001 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3061 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3121 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3181 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3241 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3301 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3361 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3421 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3481 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3541 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3601 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     3661 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3721 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3781 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     3841 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3901 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3961 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4021 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4081 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4141 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4201 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4261 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4321 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4381 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4441 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4501 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4561 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4621 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4681 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4741 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4801 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4861 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     4921 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     4981 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     5041 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     5101 cacaggaaac agctatgacc atgattacgc caaggtcgac ttaaccctag aaagataatc
     5161 atattgtgac gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc
     5221 tgtatatcga ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt
     5281 acatactaat aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa
     5341 acaaaaactc aaaatttctt ctataaagta acaaaacttt tagcagtgaa aaaaatgctt
     5401 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     5461 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     5521 tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc ctctggagat
     5581 ttagccctcc cacacataac cagagggcag caattcacga atcccaactg ccgtcggctg
     5641 tccatcactg tccttcacta tggctttgat cccaggatgc agatcgagaa gcacctgtcg
     5701 gcaccgtccg caggggctca agatgcccct gttctcattt ccgatcgcga cgatacaagt
     5761 caggttgcca gctgccgcag cagcagcagt gcccagcacc acgagttctg cacaaggtcc
     5821 cccagtaaaa tgatatacat tgacaccagt gaagatgcgg ccgtcgctag agagagctgc
     5881 gctggcgacg ctgtagtctt cagagatggg gatgctgttg attgtagccg ttgctctttc
     5941 aatgagggtg gattcttctt gagacaaagg cttggccatg gggccggggt tctcctccac
     6001 gtcgccggcc tgcttcagca ggctgaagtt ggtggcgccg ctgcccccac cgtactcgtc
     6061 aattccaagg gcatcggtaa acatctgctc aaactcgaag tcggccatat ccagagcgcc
     6121 gtagggggcg gagtcgtggg gggtaaatcc cggacccggg gaatccccgt cccccaacat
     6181 gtccagatcg aaatcgtcta gcgcgtcggc atgcgccatc gccacgtcct cgccgtctaa
     6241 gtggagctcg tcccccaggc tgacatcggt cgggggggcc gtcgacagtc tgcgcgtgtg
     6301 tcccgcgggg agaaaggaca ggcgcggagc cgccagcccc gcctcttcgg gggcgtcgtc
     6361 gtccgggaga tcgagcaggc cctcgatggt agacccgtaa ttgtttttcg tacgcgcgcg
     6421 gctgtacgcg gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc
     6481 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg
     6541 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg
     6601 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa
     6661 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc
     6721 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag
     6781 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc
     6841 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa
     6901 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc
     6961 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt
     7021 aatcacttta cttttatcta atctagacat cttatcgtca tcgtctttgt aatccatggt
     7081 ggcggatccc gcgtcacgac acctgtgttc tggcggcaaa cccgttgcga aaaagaacgt
     7141 tcacggcgac tactgcactt atatacggtt ctcccccacc ctcgggaaaa aggcggagcc
     7201 agtacacgac atcactttcc cagtttaccc cgcgccacct tctctaggca ccggttcaat
     7261 tgccgacccc tccccccaac ttctcgggga ctgtgggcga tgtgcgctct gcccactgac
     7321 gggcaccgga gccactcgag tggaatt
//
