LOCUS       sequence-483906-        6513 bp    DNA     circular SYN 20-AUG-2025
DEFINITION  Express doxycycline inducible Fully-spliced CRNDE in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6513)
  AUTHORS   Lee JS, Dan T, Zhang H, Cheng Y, Rehfeld F, Brugarolas J, Mendell JT
  TITLE     An ultraconserved snoRNA-like element in long noncoding RNA CRNDE 
            promotes ribosome biogenesis and cell proliferation.
  JOURNAL   Mol Cell. 2025 Apr 17;85(8):1543-1560.e10. doi: 
            10.1016/j.molcel.2025.03.006. Epub 2025 Apr 3.
  PUBMED    40185099
REFERENCE   2  (bases 1 to 6513)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 20, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2025.03.006"; journalName: "Mol Cell"; date: 
            "2025-04-17- 17"; volume: "85"; issue: "8"; pages: "1543-1560.e10"
COMMENT     Sequence Label: XLone-Fully-spliced CRNDE
FEATURES             Location/Qualifiers
     source          1..6513
                     /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"
     RBS             945..953
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    1475..1609
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1885..1919
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1930..1947)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1930..1946)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1939..1961)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2155..2174)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2274..2296
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2334..2352)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2420..2524
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2525..2593,2594..3385)
                     /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(2743..2762)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3556..4144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4045..4064
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4298..4315
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4432..4453
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4468..4473,4474..4491,4492..4498)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4506..4522
                     /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     4511..4533
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4530..4546
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4530..4546
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4572..4634
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4810..4891
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5008..5406)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(5407..5463)
                     /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(5473..6216)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(6217..6240)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6240..6249
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6269..6480)
                     /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 ccgccgcagc ccctggcgct aacggtcggt aacggcccgc
      421 gcgcgccgcc cgccgggggc tcgcgccagc cacgagggag cgtccgcggc ccgcgcgccc
      481 gcgcggcgga ggagaggtga gcccccgccc gggccaggcc ctctggccgc gccgtccgcc
      541 cctctagtcg tgtcccctcg tgggccgaac ggacgcggcg gtgccccgcg cccgaccaga
      601 cgtcccgtgg gctagggcct gggcctcggg ccgcgtcggc gccggtcgag gttctccggg
      661 tgtcggggtt cggggcgggc gcgcgtgggc gtggctcctc tgtccacgcc tgttcccttc
      721 gtcgccgcgg ctctcgtccg ggacacggct ttccggagta gagcccttgg aggtgttaag
      781 tgtgatgctt ccataataca tttggatgct gtcagctaag ttcacttctg aactaagggg
      841 ttcctccaaa tgttggctga aattcatccc aaggctggtc tgcaaagtct gcaattcata
      901 atggagctac tgtactggct attggaagga ggagattctg aagataagga ggtaaaacct
      961 gtttagaaat taaaaatgag ttacgattta aagaaaattc agatgactca ttgtgagtgc
     1021 tagttctctt gtaggatgcc actggaaatg ttgaaatgaa aaatattcag ccgttggtct
     1081 ttgaaatttc ctgtgatgtg tttcaatcta gatgcaaaga acatggaaaa atcaaagtgc
     1141 tcgagtggtt taaatatgtt ttgggtattc ctgtttatag actataatac ttttccaatt
     1201 aaaatcctca gttgtcacgc agaagaaggt taagctgtat ttgattgcca gttttactga
     1261 aaatgcttag tattttacag tatcaccaaa tataactagt agaccacctc ccctgcgagc
     1321 taagctggac agccaatgac gggtaagaga gtgacatttt tcactaacct aagacaggag
     1381 ggccgtcaga gctactgcct aatccaaaga cgggtaaaag tgataaaaat gtatcactcc
     1441 aacctaagac aggcgcagct tccgagggat ttgagatcca gacatgataa gatacattga
     1501 tgagtttgga caaaccaaaa ctagaatgca gtgaaaaaaa tgccttattt gtgaaatttg
     1561 tgatgctatt gccttatttg taaccattat aagctgcaat aaacaagttt gatatctata
     1621 acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata acaatataat
     1681 tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca
     1741 cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg
     1801 agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa
     1861 agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt
     1921 taagaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     1981 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     2041 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     2101 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     2161 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     2221 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     2281 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     2341 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     2401 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2461 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2521 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     2581 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     2641 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     2701 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     2761 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     2821 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     2881 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     2941 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     3001 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     3061 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     3121 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     3181 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     3241 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     3301 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     3361 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     3421 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     3481 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3541 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3601 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3661 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     3721 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3781 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3841 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3901 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3961 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     4021 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     4081 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     4141 gaaaaacgcc agcaacgcgg cctttttacg gtttctggcc ttttgctggc cttttgctca
     4201 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4261 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4321 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     4381 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     4441 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     4501 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     4561 ggtcgactta accctagaaa gataatcata ttgtgacgta cgttaaagat aatcatgcgt
     4621 aaaattgacg catgtgtttt atcggtctgt atatcgaggt ttatttatta atttgaatag
     4681 atattaagtt ttattatatt tacacttaca tactaataat aaattcaaca aacaatttat
     4741 ttatgtttat ttatttatta aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca
     4801 aaacttttag cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4861 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     4921 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     4981 tatggctgat tatgatcctc tggagattta gccctcccac acataaccag agggcagcaa
     5041 ttcacgaatc ccaactgccg tcggctgtcc atcactgtcc ttcactatgg ctttgatccc
     5101 aggatgcaga tcgagaagca cctgtcggca ccgtccgcag gggctcaaga tgcccctgtt
     5161 ctcatttccg atcgcgacga tacaagtcag gttgccagct gccgcagcag cagcagtgcc
     5221 cagcaccacg agttctgcac aaggtccccc agtaaaatga tatacattga caccagtgaa
     5281 gatgcggccg tcgctagaga gagctgcgct ggcgacgctg tagtcttcag agatggggat
     5341 gctgttgatt gtagccgttg ctctttcaat gagggtggat tcttcttgag acaaaggctt
     5401 ggccatgggg ccggggttct cctccacgtc gccggcctgc ttcagcaggc tgaagttggt
     5461 ggcgccgctg cccccgggga gcatgtcaag gtcaaaatcg tcaagagcgt cagcaggcag
     5521 catatcaagg tcaaagtcgt caagggcatc ggctgggagc atgtctaagt caaaatcgtc
     5581 aagggcgtcg gtcggcccgc cgctttcgca ctttagctgt ttctccaggc cacatatgat
     5641 tagttccagg ccgaaaagga aggcaggttc ggctccctgc cggtcgaaca gctcaattgc
     5701 ttgtttcaga agtgggggca tagaatcggt ggtaggtgtc tctctttcct cttttgctac
     5761 ttgatgctcc tgttcctcca atacgcagcc cagtgtaaag tggcccacgg cggacagagc
     5821 gtacagtgcg ttctccaggg agaagccttg ctgacacagg aacgcgagct gattttccag
     5881 ggtttcgtac tgtttctctg ttgggcgggt gccgagatgc actttagccc cgtcgcgatg
     5941 tgagaggaga gcacagcggt atgacttggc gttgttccgc agaaagtctt gccatgactc
     6001 gccttccagg gggcaggagt gggtatgatg cctgtccagc atctcgattg gcagggcatc
     6061 gagcagggcc cgcttgttct tcacgtgcca gtacagggta ggctgctcaa ctcccagctt
     6121 ttgagcgagt ttccttgtcg tcaggccttc gataccgact ccattgagta attccagagc
     6181 agagtttatg actttgctct tgtccagtct agacatctta tcgtcatcgt ctttgtaatc
     6241 catggtggcg gatcccgcgt cacgacacct gtgttctggc ggcaaacccg ttgcgaaaaa
     6301 gaacgttcac ggcgactact gcacttatat acggttctcc cccaccctcg ggaaaaaggc
     6361 ggagccagta cacgacatca ctttcccagt ttaccccgcg ccaccttctc taggcaccgg
     6421 ttcaattgcc gacccctccc cccaacttct cggggactgt gggcgatgtg cgctctgccc
     6481 actgacgggc accggagcca ctcgagtgga att
//
