LOCUS       sequence-327919-        7281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SIM mutant tagged with mCherry and eDHFR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7281)
  AUTHORS   Zhang H, Zhao R, Tones J, Liu M, Dilley RL, Chenoweth DM, Greenberg 
            RA, Lampson MA
  TITLE     Nuclear body phase separation drives telomere clustering in ALT 
            cancer cells.
  JOURNAL   Mol Biol Cell. 2020 Aug 15;31(18):2048-2056. doi: 
            10.1091/mbc.E19-10-0589. Epub 2020 Jun 24.
  PUBMED    32579423
REFERENCE   2  (bases 1 to 7281)
  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: 
            "10.1091/mbc.E19-10-0589"; journalName: "Mol Biol Cell"; date: 
            "2020-08-15- 15"; volume: "31"; issue: "18"; pages: "2048-2056"
COMMENT     Sequence Label: mCherry-eDHFR-SIMvada
FEATURES             Location/Qualifiers
     source          1..7281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     163..185
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     247..266
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             322..1029
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(467..485)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     735..754
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1048..1521
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             1522..1551
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1721..1740)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1786..1841
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1840..1859)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    2205..2238
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2251..2372
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2288..2307)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2342..2361
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2391..2411)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2391..2409)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2430..2446)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2430..2446)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2443..2465)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2454..2470
                     /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)."
     promoter        complement(join(2478..2484,2485..2502,2503..2508))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2523..2544
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2661..2678)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2832..3420)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2912..2931)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3591..4382,4383..4451))
                     /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     4214..4233
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4452..4556)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    4629..4677
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    4889..4922
                     /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)."
     CDS             4927..5526
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4927..4946)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5423..5443
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5662..5679)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5668..5779
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        5924..6227
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6229..6503
                     /label=chicken beta-actin promoter
     intron          join(6504..7281,1..239)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
ORIGIN
        1 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
       61 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
      121 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
      181 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
      241 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgctcgacc
      301 accggcgcgc caccggtcac catggtgagc aagggcgagg aggataacat ggccatcatc
      361 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
      421 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
      481 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
      541 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
      601 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
      661 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
      721 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag
      781 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
      841 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag
      901 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
      961 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
     1021 ctgtataagg ccggcctgga gtctagaatc agtctgattg cggcgttagc ggtagatcgc
     1081 gttatcggca tggaaaacgc catgccgtgg aacctgcctg ccgatctcgc ctggtttaaa
     1141 cgcaacacct taaataaacc cgtgattatg ggccgccata cctgggaatc aatcggtcgt
     1201 ccgttgccag gacgcaaaaa tattatcctc agcagtcaac cgggtacgga cgatcgcgta
     1261 acgtgggtga agtcggtgga tgaagccatc gcggcgtgtg gtgacgtacc agaaatcatg
     1321 gtgattggcg gcggtcgcgt ttatgaacag ttcttgccaa aagcgcaaaa actgtatctg
     1381 acgcatatcg acgcagaagt ggaaggcgac acccatttcc cggattacga gccggatgac
     1441 tgggaatcgg tattcagcga attccacgat gctgatgcgc agaactctca cagctattgc
     1501 tttgagattc tggagcggcg ggaacaaaaa ctcatctcag aagaggatct taacaaagtc
     1561 gatgtagccg acgccacgat cgaatctagc agcgatgaag aagaagatcc accggctaaa
     1621 cgtggatcct agggccgcaa ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg
     1681 ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa
     1741 attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta
     1801 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga
     1861 gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc
     1921 atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc
     1981 cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta
     2041 tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac
     2101 tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat
     2161 ggagatccct cgacctgcac cgtcgagcta gcatatggat ccatataact tcgtataatg
     2221 tatgctatac gaagttatta cgataccgtc aacttgttta ttgcagctta taatggttac
     2281 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     2341 tgtggtttgt ccaaactcat caatgtatct taggtaccca gcttttgttc cctttagtga
     2401 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     2461 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     2521 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2581 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2641 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     2701 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     2761 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2821 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2881 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     2941 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3001 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3061 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3121 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3181 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3241 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3301 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3361 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3421 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     3481 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     3541 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     3601 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     3661 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     3721 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     3781 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3841 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3901 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3961 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4021 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4081 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4141 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4201 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4261 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4321 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4381 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4441 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4501 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     4561 tttccccgaa aagtgccaag cttaataaac acggaaggag acaataccgg aaggaacccg
     4621 cgctatgacg gcaataaaaa gacagaataa aacgcacggg tgttgggtcg tttgttcata
     4681 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     4741 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     4801 cagggctcgc agccaacgtc ggggcggcag gccctgccat agccactggc cccgtgggtt
     4861 agggacgggg tcccccattc gagaataaat aacttcgtat aaagtatcct atacgaagtt
     4921 atcaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg
     4981 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac
     5041 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     5101 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     5161 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     5221 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     5281 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt
     5341 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     5401 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     5461 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     5521 gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatggc
     5581 tccgaccgaa gccacccggg gcggccccgc cgaccccgca cccgcccccg aggcccaccg
     5641 actctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     5701 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     5761 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     5821 tggggcagga cagcaagggg gaggattggg aagacaatag cactagttat taatagtaat
     5881 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     5941 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     6001 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     6061 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     6121 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     6181 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
     6241 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
     6301 ttatttttta attattttgt gcagcgatgg gggcgggggg ggggggggcc gcgccaggcg
     6361 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
     6421 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     6481 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     6541 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     6601 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg
     6661 tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg
     6721 ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg
     6781 cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg
     6841 tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga
     6901 acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg
     6961 tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg
     7021 gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc
     7081 aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc
     7141 gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt
     7201 tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga
     7261 aatctgggag gcgccgccgc a
//
