LOCUS       sequence-484001-        7520 bp    DNA     circular SYN 20-AUG-2025
DEFINITION  Express doxycycline inducible intron fragment upstream of UCE of 
            CRNDE in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7520)
  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 7520)
  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 upstream
FEATURES             Location/Qualifiers
     source          1..7520
                     /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    2482..2616
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2892..2926
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2937..2954)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2937..2953)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2946..2968)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3162..3181)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3281..3303
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3341..3359)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3427..3531
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3532..3600,3601..4392)
                     /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(3750..3769)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4563..5151
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5052..5071
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5305..5322
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5439..5460
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5475..5480,5481..5498,5499..5505)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5513..5529
                     /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     5518..5540
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5537..5553
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5537..5553
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5579..5641
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5817..5898
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6015..6413)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(6414..6470)
                     /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(6480..7223)
                     /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(7224..7247)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7247..7256
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7276..7487)
                     /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 gactagtaga ccacctcccc tgcgagctaa gctggacagc
     2341 caatgacggg taagagagtg acatttttca ctaacctaag acaggagggc cgtcagagct
     2401 actgcctaat ccaaagacgg gtaaaagtga taaaaatgta tcactccaac ctaagacagg
     2461 cgcagcttcc gagggatttg agatccagac atgataagat acattgatga gtttggacaa
     2521 accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga tgctattgcc
     2581 ttatttgtaa ccattataag ctgcaataaa caagtttgat atctataaca agaaaatata
     2641 tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt
     2701 aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata
     2761 gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc
     2821 gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata
     2881 tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa gaattcactg
     2941 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     3001 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     3061 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg
     3121 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     3181 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3241 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3301 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     3361 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     3421 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3481 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3541 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3601 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3661 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3721 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     3781 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3841 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3901 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3961 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4021 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     4081 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4141 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4201 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     4261 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4321 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4381 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4441 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4501 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4561 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4621 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4681 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     4741 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4801 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4861 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4921 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4981 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5041 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5101 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5161 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     5221 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     5281 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     5341 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     5401 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     5461 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     5521 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaaggt cgacttaacc
     5581 ctagaaagat aatcatattg tgacgtacgt taaagataat catgcgtaaa attgacgcat
     5641 gtgttttatc ggtctgtata tcgaggttta tttattaatt tgaatagata ttaagtttta
     5701 ttatatttac acttacatac taataataaa ttcaacaaac aatttattta tgtttattta
     5761 tttattaaaa aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa cttttagcag
     5821 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     5881 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     5941 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat
     6001 gatcctctgg agatttagcc ctcccacaca taaccagagg gcagcaattc acgaatccca
     6061 actgccgtcg gctgtccatc actgtccttc actatggctt tgatcccagg atgcagatcg
     6121 agaagcacct gtcggcaccg tccgcagggg ctcaagatgc ccctgttctc atttccgatc
     6181 gcgacgatac aagtcaggtt gccagctgcc gcagcagcag cagtgcccag caccacgagt
     6241 tctgcacaag gtcccccagt aaaatgatat acattgacac cagtgaagat gcggccgtcg
     6301 ctagagagag ctgcgctggc gacgctgtag tcttcagaga tggggatgct gttgattgta
     6361 gccgttgctc tttcaatgag ggtggattct tcttgagaca aaggcttggc catggggccg
     6421 gggttctcct ccacgtcgcc ggcctgcttc agcaggctga agttggtggc gccgctgccc
     6481 ccggggagca tgtcaaggtc aaaatcgtca agagcgtcag caggcagcat atcaaggtca
     6541 aagtcgtcaa gggcatcggc tgggagcatg tctaagtcaa aatcgtcaag ggcgtcggtc
     6601 ggcccgccgc tttcgcactt tagctgtttc tccaggccac atatgattag ttccaggccg
     6661 aaaaggaagg caggttcggc tccctgccgg tcgaacagct caattgcttg tttcagaagt
     6721 gggggcatag aatcggtggt aggtgtctct ctttcctctt ttgctacttg atgctcctgt
     6781 tcctccaata cgcagcccag tgtaaagtgg cccacggcgg acagagcgta cagtgcgttc
     6841 tccagggaga agccttgctg acacaggaac gcgagctgat tttccagggt ttcgtactgt
     6901 ttctctgttg ggcgggtgcc gagatgcact ttagccccgt cgcgatgtga gaggagagca
     6961 cagcggtatg acttggcgtt gttccgcaga aagtcttgcc atgactcgcc ttccaggggg
     7021 caggagtggg tatgatgcct gtccagcatc tcgattggca gggcatcgag cagggcccgc
     7081 ttgttcttca cgtgccagta cagggtaggc tgctcaactc ccagcttttg agcgagtttc
     7141 cttgtcgtca ggccttcgat accgactcca ttgagtaatt ccagagcaga gtttatgact
     7201 ttgctcttgt ccagtctaga catcttatcg tcatcgtctt tgtaatccat ggtggcggat
     7261 cccgcgtcac gacacctgtg ttctggcggc aaacccgttg cgaaaaagaa cgttcacggc
     7321 gactactgca cttatatacg gttctccccc accctcggga aaaaggcgga gccagtacac
     7381 gacatcactt tcccagttta ccccgcgcca ccttctctag gcaccggttc aattgccgac
     7441 ccctcccccc aacttctcgg ggactgtggg cgatgtgcgc tctgcccact gacgggcacc
     7501 ggagccactc gagtggaatt
//
