LOCUS       sequence-466560-        5125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone plasmid used to clone lncRNA inserts in preparation for 
            RMCE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5125)
  AUTHORS   Trotman JB, Abrash EW, Murvin MM, Braceros AK, Li S, Boyson SP, 
            Salcido RT, Cherney RE, Bischoff SR, Kaufmann K, Eberhard QE, Zhang 
            Z, Cowley DO, Calabrese JM
  TITLE     Isogenic comparison of Airn and Xist reveals core principles of 
            Polycomb recruitment by lncRNAs.
  JOURNAL   Mol Cell. 2025 Mar 20;85(6):1117-1133.e14. doi: 
            10.1016/j.molcel.2025.02.014.
  PUBMED    40118040
REFERENCE   2  (bases 1 to 5125)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2025.02"; journalName: "Mol Cell"; date: 
            "2025-03-20- 20"; volume: "85"; issue: "6"; pages: "1117-1133.e14"
COMMENT     Sequence Label: pCARGO-RMCE
FEATURES             Location/Qualifiers
     source          1..5125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(158..471)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     primer_bind     complement(160..184)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    231..249
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    267..285
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    302..320
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    338..356
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    374..392
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    409..427
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    445..463
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    complement(481..514)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        554..658
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(659..727,728..1519)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(877..896)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1690..2278
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2179..2198
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2432..2449
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2464..2604
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2530..2549)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2690..2712
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2706..2897)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3174..3196
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3193..3209
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3193..3209
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3230..3248
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(3281..3314)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(3320..3353)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(3361..4386)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(4405..4452)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(4464..4963)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4512..4531)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     4905..4928
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    complement(4976..5009)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    5028..5109
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5053..5072
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
       61 tctacaaatg tggtatggct gattatgatc ctctagagat ttaaattgat atcgtcgaca
      121 agcttatcga tgcggccgcg ctagcacgcg tggatccgcg atctgacggt tcactaaacg
      181 agctctgctt atataggcct cccaccgtac acgcctacct cgacatacgt tctctatcac
      241 tgatagggag taaactcgac atacgttctc tatcactgat agggataaac tcgacatacg
      301 ttctctatca ctgataggga gtaaactcga catacgttct ctatcactga tagggagtaa
      361 actcgacata cgttctctat cactgatagg gagtaaactc gacatcgttc tctatcactg
      421 atagggagta aactcgacat acgttctcta tcactgatag ggagtaaact cgggcgcgcc
      481 taccgttcgt atagcataca ttatacgaag ttatcctgca gggacgtcag gtggcacttt
      541 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      601 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
      661 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
      721 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
      781 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
      841 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      901 tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      961 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     1021 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     1081 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     1141 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     1201 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     1261 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     1321 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     1381 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     1441 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     1501 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     1561 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1621 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1681 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1741 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1801 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     1861 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1921 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1981 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     2041 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     2101 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2161 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2221 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2281 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2341 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2401 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     2461 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
     2521 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     2581 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     2641 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     2701 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     2761 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     2821 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     2881 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     2941 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     3001 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     3061 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     3121 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga ctgagcggat
     3181 aacaatttca cacaggaaac agctatgacc atgattacgc caagctcgaa attaaccctc
     3241 actaaaggga acaaaagctg gtaccgggcc ccccctcgag ataacttcgt ataggatact
     3301 ttatacgaag ttatcttaag aagttcctat actttctaga gaataggaac ttctgtacat
     3361 ctattccttt gccctcggac gagtgctggg gcgtcggttt ccactatcgg cgagtacttc
     3421 tacacagcca tcggtccaga cggccgcgct tctgcgggcg atttgtgtac gcccgacagt
     3481 cccggctccg gatcggacga ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa
     3541 attgccgtca accaagctct gatagagttg gtcaagacca atgcggagca tatacgcccg
     3601 gagccgcggc gatcctgcaa gctccggatg cctccgctcg aagtagcgcg tctgctgctc
     3661 catacaagcc aaccacggcc tccagaagaa gatgttggcg acctcgtatt gggaatcccc
     3721 gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg ccattgtccg tcaggacatt
     3781 gttggagccg aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct cggcccaaag
     3841 catcagctca tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg
     3901 ccagtgatac acatggggat cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg
     3961 accgattcct tgcggtccga atgggccgaa cccgctcgtc tggctaagat cggccgcagc
     4021 gatcgcatcc atggcctccg cgaccggctg cagaacagcg ggcagttcgg tttcaggcag
     4081 gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa taggtcaggc tctcgctgaa
     4141 ctccccaatg tcaagcactt ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac
     4201 ataacgatct ttgtagaaac catcggcgca gctatttacc cgcaggacat atccacgccc
     4261 tcctacatcg aagctgaaag cacgagattc ttcgccctcc gagagctgca tcaggtcgga
     4321 gacgctgtcg aacttttcga tcagaaactt ctcgacagat gtcgcggtga gttcaggctt
     4381 tttcatggtt tagttcctca ccttgtcgta ttatactatg ccgatatact atgccgatga
     4441 ttaattgtca acaggctgca ggtcgaaagg cccggagatg aggaagagga gaacagcgcg
     4501 gcagacgtgc gcttttgaag cgtgcagaat gccgggcctc cggaggacct tcgggcgccc
     4561 gccccgcccc tgagcccgcc cctgagcccg cccccggacc caccccttcc cagcctctga
     4621 gcccagaaag cgaaggagca aagctgctat tggccgctgc cccaaaggcc tacccgcttc
     4681 cattgctcag cggtgctgtc catctgcacg agactagtga gacgtgctac ttccatttgt
     4741 cacgtcctgc acgacgcgag ctgcggggcg ggggggaact tcctgactag gggaggagta
     4801 gaaggtggcg cgaaggggcc accaaagaac ggagccggtt ggcgcctacc ggtggatgtg
     4861 gaatgtgtgc gaggccagag gccacttgtg tagcgccaag tgcccagcgg ggctgctaaa
     4921 gcgcatgctc cagactgcct tgggaaaagc gcctccccta cccggtagaa tgcatgaagt
     4981 tcctatactt tctagagaat aggaacttcg gaattcagat cttcgaggca gtgaaaaaaa
     5041 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     5101 aaacaagtta acaacaacaa ttgca
//
