LOCUS       sequence-327909-        7280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tag SIM with mCherry and eDHFR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7280)
  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 7280)
  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_SIM#1
FEATURES             Location/Qualifiers
     source          1..7280
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     446..468
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     530..549
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             605..1312
                     /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(750..768)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1018..1037
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1331..1804
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             1805..1834
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2003..2022)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2068..2123
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2122..2141)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    2487..2520
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2533..2654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2570..2589)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2624..2643
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2673..2693)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2673..2691)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2712..2728)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2712..2728)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2725..2747)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2736..2752
                     /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(2760..2766,2767..2784,2785..2790))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2805..2826
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2943..2960)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3114..3702)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3194..3213)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3873..4664,4665..4733))
                     /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     4496..4515
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4734..4838)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    4911..4959
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    5171..5204
                     /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             5209..5808
                     /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(5209..5228)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5705..5725
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5944..5961)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5950..6061
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        6206..6509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          join(6786..7280,1..522)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
ORIGIN
        1 gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc
       61 gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc
      121 ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg
      181 gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg
      241 tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc
      301 ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg
      361 ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc
      421 cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga
      481 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg
      541 gttattgtgc tgtctcatca ttttggcaaa gaattgctcg accaccggcg cgccaccggt
      601 caccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
      661 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
      721 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
      781 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
      841 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
      901 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
      961 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     1021 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     1081 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     1141 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     1201 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     1261 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtata aggccggcct
     1321 ggagtctaga atcagtctga ttgcggcgtt agcggtagat cgcgttatcg gcatggaaaa
     1381 cgccatgccg tggaacctgc ctgccgatct cgcctggttt aaacgcaaca ccttaaataa
     1441 acccgtgatt atgggccgcc atacctggga atcaatcggt cgtccgttgc caggacgcaa
     1501 aaatattatc ctcagcagtc aaccgggtac ggacgatcgc gtaacgtggg tgaagtcggt
     1561 ggatgaagcc atcgcggcgt gtggtgacgt accagaaatc atggtgattg gcggcggtcg
     1621 cgtttatgaa cagttcttgc caaaagcgca aaaactgtat ctgacgcata tcgacgcaga
     1681 agtggaaggc gacacccatt tcccggatta cgagccggat gactgggaat cggtattcag
     1741 cgaattccac gatgctgatg cgcagaactc tcacagctat tgctttgaga ttctggagcg
     1801 gcgggaacaa aaactcatct cagaagagga tcttaagagc aaagtagatg tgatcgactt
     1861 gaccatcgaa agcagcagcg acgaagaaga agatccgcct gccaaacgct aataggccgc
     1921 aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     1981 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     2041 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     2101 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     2161 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     2221 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     2281 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     2341 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     2401 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc
     2461 accgtcgagc tagcatatgg atccatataa cttcgtataa tgtatgctat acgaagttat
     2521 tacgataccg tcaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     2581 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     2641 atcaatgtat cttaggtacc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
     2701 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2761 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2821 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2881 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2941 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3001 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3061 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3121 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3181 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3241 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3301 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3361 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3421 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3481 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3541 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3601 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3661 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3721 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3781 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3841 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3901 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3961 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     4021 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4081 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4141 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     4201 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4261 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4321 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     4381 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4441 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4501 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4561 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4621 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4681 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4741 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4801 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4861 agcttaataa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     4921 aagacagaat aaaacgcacg ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     4981 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     5041 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     5101 tcggggcggc aggccctgcc atagccactg gccccgtggg ttagggacgg ggtcccccat
     5161 tcgagaataa ataacttcgt ataaagtatc ctatacgaag ttatcaccat gaccgagtac
     5221 aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg caccctcgcc
     5281 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg ccacatcgag
     5341 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     5401 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     5461 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     5521 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     5581 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     5641 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     5701 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     5761 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac
     5821 gacccgcagc gcccgaccga aaggagcgca cgaccccatg gctccgaccg aagccacccg
     5881 gggcggcccc gccgaccccg cacccgcccc cgaggcccac cgactctaga gctcgctgat
     5941 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     6001 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     6061 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     6121 gggaggattg ggaagacaat agcactagtt attaatagta atcaattacg gggtcattag
     6181 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     6241 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     6301 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     6361 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     6421 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     6481 tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc
     6541 tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt
     6601 gtgcagcgat gggggcgggg ggggggnnnn nnncgccagg cggggcgggg cggggcgagg
     6661 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
     6721 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
     6781 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc
     6841 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
     6901 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc
     6961 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg
     7021 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg
     7081 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg
     7141 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg
     7201 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc
     7261 tgcacccccc tccccgagtt
//
