LOCUS       sequence-483917-        8119 bp    DNA     circular SYN 20-AUG-2025
DEFINITION  Express doxycycline inducible UCE intron 3' deletion of CRNDE in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8119)
  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 8119)
  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-UCE intron 3' deletion
FEATURES             Location/Qualifiers
     source          1..8119
                     /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             442..450
                     /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    3081..3215
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   3491..3525
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3536..3553)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3536..3552)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3545..3567)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3761..3780)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3880..3902
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3940..3958)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4026..4130
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4131..4199,4200..4991)
                     /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(4349..4368)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5162..5750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5651..5670
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5904..5921
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6038..6059
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6074..6079,6080..6097,6098..6104)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6112..6128
                     /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     6117..6139
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6136..6152
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6136..6152
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   6178..6240
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    6416..6497
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6614..7012)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(7013..7069)
                     /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(7079..7822)
                     /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(7823..7846)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7846..7855
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7875..8086)
                     /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 ccagtctgca attcataatg gagctactgt actggctatt
      421 ggaaggagga gattctgaag ataaggaggt aatattatct cttttaaaag aatactttcc
      481 tctgtaatcc tgaatcttta ttacatgtaa gaactttgtg cagtagacag caatttcttt
      541 gaatttggta tatggaaaca attttatttt cctctgctaa gtttttgagc ctgcctcttc
      601 tagtgccatg gactgcattg gtagagctga gaaatatcat ttagccatac tcagcaccct
      661 taaaatagct tctttctgag aattagatct gtgaaggtgt cctgcacagt tcttgtagat
      721 gtcattttag tttgtggttg acgtgcatgc atttagcatg ttgcttaacc gtcctcattc
      781 gcctcccagt tctttgttgc cttcatttgg ggggatgtgt tttctgctgg atgatttact
      841 gctagacatg accaaactct gagtataaga cttggtgttt ggcagctggt tccagtgctc
      901 tgctgagaag tagttgggcc cagcctgggg cattgtagtg tcctggaggc cgggagctcc
      961 tgcacagggg ttcttttcct ggttcagagc cttaggtgcc tttccacttt tcacgtaatc
     1021 ttttcctagg ttggcttgct gcttactttg cagctgttgc agggattcac atggaatgga
     1081 gggctccctt ttattgtgga ttatttcttt gataatttac ccatgtgctt tttcattttc
     1141 aaaaacccag tggtgtttaa gaatgaaaga ttcttgaaga acaaaaattg ggttaaattt
     1201 gtatctatca gaaaagattc tatcctctga tgctatgtag gcacattgaa tatgcaggct
     1261 aaattaaaaa cagagtaaaa cctttttata atatgccaat tactatatct aagaatgttt
     1321 atacaggctg aaattttgta atggtattta tatctgtttt cctttttaag gaaaataata
     1381 tacttttcta ggcatcaaaa atatctgccc ctttcactaa tgctgatgta ccaccttccc
     1441 ccaaccccca acgctaaatt tgactggctt aaaaacatct gccccctgaa ctatatctgg
     1501 ggaggaatat taataaaaca atgaaggact tcatggtgtc tagttatata attaggaaat
     1561 tggttagaaa cgtggctaaa accagtttct ttcattaaaa gaactagtgt acatttaaaa
     1621 acaaaaacct tcaggaaaaa gactctttga tctttaagtc aaatagtatt tttggttaac
     1681 acagcaggaa ggggaaatat accaatttca gattctttta tttatgccta agtagaggtt
     1741 gtaagcagct aaggaagtga ttaaaatgta gtctagtaaa aaatggtgct gattacttgg
     1801 aaagcagttt acatttgcaa aaaaattcag tattatgacc ttcaccaact tttacacatc
     1861 atatacttgg gttattactt atggtatagc ttgtgtatgg aatgcaaggg tattttcaaa
     1921 ttgactaggt cttgctggtc atcttaaaat atgttacaat attacaaaat tgaaatgtaa
     1981 tattttttaa tagaaggaaa atataaattt aatatctggg caattgagac ctttaaactt
     2041 actttaaaag tatgatcttg atgtatatga tactgttttg tctttgctat attaacagaa
     2101 ttagaggggt gttctgcaat tcaaatacct tatatattcc aaattttatt ctctataatg
     2161 gacttttaaa ataaaaggta tatgtgcttc aagagggcaa aatttgaatc atgagctaat
     2221 ttgctaagca tcagattata gaaaagcatc cttgattaat ttggaactgt gaaagggggc
     2281 gggtaaaact gttttctgca gaaatttact agtgcagcaa ccatttaaat taaatgtttg
     2341 ttaacataat agtgatggca ttttctcctc cccctccttg tggttttgtc caactagatg
     2401 ttacagtggc agttgcactg actgttaagt gtttaaatga tgacaccatt atgtgaagtg
     2461 attttgaaat gagagattcc agccaagaat tacatctgct cccatctcct tcaaatcata
     2521 ctctctggca gtacagatta tgattgattt gtttgtgaca gattgcagga aacagtcatt
     2581 gatttttcaa tattttacct taaaattatt tacagttgta accatgggga ggtattttca
     2641 tgggctgtca gcccctgaaa gactaggata atattccctg ctctctgaca agacaaatta
     2701 cctgtaatga gtgcagtagc tgaagggtat acttttattt taaaatatgt caataacccc
     2761 agtgactaaa cgaatattga tttagcataa tgaagcctga gtaacgtgaa aatgagcttt
     2821 ttcaaggggc atggtaaagt ctttcttttt agctggttgt aagaagcttt tgattctttt
     2881 cagccagctg gtaggaatat actagtagac cacctcccct gcgagctaag ctggacagcc
     2941 aatgacgggt aagagagtga catttttcac taacctaaga caggagggcc gtcagagcta
     3001 ctgcctaatc caaagacggg taaaagtgat aaaaatgtat cactccaacc taagacaggc
     3061 gcagcttccg agggatttga gatccagaca tgataagata cattgatgag tttggacaaa
     3121 ccaaaactag aatgcagtga aaaaaatgcc ttatttgtga aatttgtgat gctattgcct
     3181 tatttgtaac cattataagc tgcaataaac aagtttgata tctataacaa gaaaatatat
     3241 atataataag ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta
     3301 aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag
     3361 ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg
     3421 actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat
     3481 ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaag aattcactgg
     3541 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     3601 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     3661 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
     3721 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
     3781 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3841 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3901 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     3961 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     4021 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4081 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4141 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4201 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4261 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4321 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4381 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4441 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4501 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4561 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4621 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4681 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4741 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4801 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     4861 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4921 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4981 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     5041 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5101 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5161 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5221 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5281 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5341 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5401 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5461 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5521 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5581 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5641 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5701 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5761 acgcggcctt tttacggttt ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5821 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5881 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     5941 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     6001 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     6061 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     6121 gataacaatt tcacacagga aacagctatg accatgatta cgccaaggtc gacttaaccc
     6181 tagaaagata atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg
     6241 tgttttatcg gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat
     6301 tatatttaca cttacatact aataataaat tcaacaaaca atttatttat gtttatttat
     6361 ttattaaaaa aaaacaaaaa ctcaaaattt cttctataaa gtaacaaaac ttttagcagt
     6421 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     6481 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     6541 aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg
     6601 atcctctgga gatttagccc tcccacacat aaccagaggg cagcaattca cgaatcccaa
     6661 ctgccgtcgg ctgtccatca ctgtccttca ctatggcttt gatcccagga tgcagatcga
     6721 gaagcacctg tcggcaccgt ccgcaggggc tcaagatgcc cctgttctca tttccgatcg
     6781 cgacgataca agtcaggttg ccagctgccg cagcagcagc agtgcccagc accacgagtt
     6841 ctgcacaagg tcccccagta aaatgatata cattgacacc agtgaagatg cggccgtcgc
     6901 tagagagagc tgcgctggcg acgctgtagt cttcagagat ggggatgctg ttgattgtag
     6961 ccgttgctct ttcaatgagg gtggattctt cttgagacaa aggcttggcc atggggccgg
     7021 ggttctcctc cacgtcgccg gcctgcttca gcaggctgaa gttggtggcg ccgctgcccc
     7081 cggggagcat gtcaaggtca aaatcgtcaa gagcgtcagc aggcagcata tcaaggtcaa
     7141 agtcgtcaag ggcatcggct gggagcatgt ctaagtcaaa atcgtcaagg gcgtcggtcg
     7201 gcccgccgct ttcgcacttt agctgtttct ccaggccaca tatgattagt tccaggccga
     7261 aaaggaaggc aggttcggct ccctgccggt cgaacagctc aattgcttgt ttcagaagtg
     7321 ggggcataga atcggtggta ggtgtctctc tttcctcttt tgctacttga tgctcctgtt
     7381 cctccaatac gcagcccagt gtaaagtggc ccacggcgga cagagcgtac agtgcgttct
     7441 ccagggagaa gccttgctga cacaggaacg cgagctgatt ttccagggtt tcgtactgtt
     7501 tctctgttgg gcgggtgccg agatgcactt tagccccgtc gcgatgtgag aggagagcac
     7561 agcggtatga cttggcgttg ttccgcagaa agtcttgcca tgactcgcct tccagggggc
     7621 aggagtgggt atgatgcctg tccagcatct cgattggcag ggcatcgagc agggcccgct
     7681 tgttcttcac gtgccagtac agggtaggct gctcaactcc cagcttttga gcgagtttcc
     7741 ttgtcgtcag gccttcgata ccgactccat tgagtaattc cagagcagag tttatgactt
     7801 tgctcttgtc cagtctagac atcttatcgt catcgtcttt gtaatccatg gtggcggatc
     7861 ccgcgtcacg acacctgtgt tctggcggca aacccgttgc gaaaaagaac gttcacggcg
     7921 actactgcac ttatatacgg ttctccccca ccctcgggaa aaaggcggag ccagtacacg
     7981 acatcacttt cccagtttac cccgcgccac cttctctagg caccggttca attgccgacc
     8041 cctcccccca acttctcggg gactgtgggc gatgtgcgct ctgcccactg acgggcaccg
     8101 gagccactcg agtggaatt
//
