LOCUS       sequence-307154-        3454 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Fluorescent reporter for intestinal stem cells through ASCL2 
            transcriptional activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 3454)
  AUTHORS   Heinz MC, Oost KC, Snippert HJG
  TITLE     Introducing the Stem Cell ASCL2 Reporter STAR into Intestinal 
            Organoids.
  JOURNAL   STAR Protoc. 2020 Oct 8;1(3):100126. doi: 
            10.1016/j.xpro.2020.100126. eCollection 2020 Dec 18.
  PUBMED    33377020
REFERENCE   2  (bases 1 to 3454)
  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"; journalName: "STAR 
            Protoc."; date: "2020-10-8"; volume: "1(3):100126. doi"; pages: " 
            10.1016/j.xpro.2020.100126. eCollection 2020 Dec 18"
COMMENT     Sequence Label: Tol2 8xSTAR-mScarletI-NLS. PGK-puro
FEATURES             Location/Qualifiers
     source          1..3454
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             829..1524
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(962..980)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1230..1249
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1531..1551
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1590..1724
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        1802..2301
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1837..1860)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2234..2253
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2313..2360
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2409..3008
                     /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(2409..2428)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2905..2925
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3037..3171
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(3265..3427,3428..3439)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 agaggtgtaa agtacttgag taattttact tgattactgt acttaagtat tatttttggg
       61 gatttttact ttacttgagt acaattaaaa atcaatactt ttacttttac ttaattacat
      121 ttttttagaa aaaaaagtac tttttactcc ttacaatttt atttacagtc aaaaagtact
      181 tattttttgg agatcacttc attctatttt cccttgctat taccaaacca attgaattgc
      241 gctgatgccc agtttaattt aaatagatct atcgatcggt acccctaggg tgaaaaggca
      301 ggtgcaaagc aaatcctatc ccagcttcta gctaggtgaa aaggcaggtg caaagcaaat
      361 cctatcccag cttctagcta gggtgaaaag gcaggtgcaa agcaaatcct atcccagctt
      421 ctagctaggt gaaaaggcag gtgcaaagca aatcctatcc cagcttctag ctagggtgaa
      481 aaggcaggtg caaagcaaat cctatcccag cttctagcta ggtgaaaagg caggtgcaaa
      541 gcaaatccta tcccagcttc tagctagggt gaaaaggcag gtgcaaagca aatcctatcc
      601 cagcttctag ctaggtgaaa aggcaggtgc aaagcaaatc ctatcccagc ttctagctag
      661 cggcgcgccg gatccggagg ggacaagtgg agggaaagtg gggccgggcg gggggtgcct
      721 gggaagccag gctgcgctga cgtcactggg cgcgcaattc gggctggagc gctttaaaaa
      781 acgagcgtgc aagcagagag gctgctccac accgctcagg ccgcgaccat ggtgagcaag
      841 ggcgaggcag tgatcaagga gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac
      901 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
      961 gccaagctga aggtgaccaa gggtggcccc ctgcccttct cctgggacat cctgtcccct
     1021 cagttcatgt acggctccag ggccttcatc aagcaccccg ccgacatccc cgactactat
     1081 aagcagtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     1141 gccgtgaccg tgacccagga cacctccctg gaggacggca ccctgatcta caaggtgaag
     1201 ctccgcggca ccaacttccc tcctgacggc cccgtaatgc agaagaagac aatgggctgg
     1261 gaagcgtcca ccgagcggtt gtaccccgag gacggcgtgc tgaagggcga cattaagatg
     1321 gccctgcgcc tgaaggacgg cggacgctac ctggcggact tcaagaccac ctacaaggcc
     1381 aagaagcccg tgcagatgcc cggcgcctac aacgtggacc gcaagttgga catcacctcc
     1441 cacaacgagg actacaccgt ggtggaacag tacgaacgct ccgagggccg ccactccacc
     1501 ggcggcatgg acgagctgta caagtccagg ccaaaaaaga aaagaaaggt catcgattag
     1561 tctagaacta tagtgagtcg tattacgtag atccagacat gataagatac attgatgagt
     1621 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     1681 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     1741 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaattc gcggccgcac
     1801 cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg
     1861 ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg
     1921 cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc
     1981 aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca
     2041 cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt
     2101 tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg
     2161 ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc
     2221 tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct
     2281 tcctcatctc cgggcctttc gacctgcagc ctgttgacaa ttaatcatcg gcatagtata
     2341 tcggcatagt ataatacgac aaggtgagga actaaaccgt cgacgatttc actagtctcg
     2401 aagccaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     2461 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     2521 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     2581 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     2641 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     2701 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     2761 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     2821 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     2881 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     2941 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     3001 gtgcctgact agaactatag tgagtcgtat tacgtagatc cagacatgat aagatacatt
     3061 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     3121 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     3181 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaattaagc
     3241 ttaaacaaga atctctagtt ttctttcttg cttttacttt tacttcctta atactcaagt
     3301 acaattttaa tggagtactt ttttactttt actcaagtaa gattctagcc agatactttt
     3361 acttttaatt gagtaaaatt ttccctaagt acttgtactt tcacttgagt aaaatttttg
     3421 agtacttttt acacctctgc tcgaccatat ggga
//
