LOCUS       sequence-312911-        6392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for intestinal stem cells through ASCL2 
            transcriptional activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6392)
  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 6392)
  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-sTomato-NLS. PGK-puro
FEATURES             Location/Qualifiers
     source          1..6392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(413..600,601..612)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     promoter        complement(625..643)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(650..667)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(650..666)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(659..681)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      807..1262
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(894..913)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1104..1125
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1340..1444
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1445..1513,1514..2305)
                     /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(1663..1682)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2476..3064
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2965..2984
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3218..3235
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3352..3373
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3388..3393,3394..3411,3412..3418)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3426..3442
                     /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     3431..3453
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3450..3466
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3450..3466
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3484..3502
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3484..3501
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    join(3553..3564,3565..3727)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    3821..3955
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(3984..4583)
                     /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(4067..4087)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4564..4583
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4632..4679)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(4691..5190)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4739..4758)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     5132..5155
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     polyA_signal    5268..5402
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5441..5461)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5557..5580)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(5755..5774)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     6024..6042
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
ORIGIN
        1 gctagaagct gggataggat ttgctttgca cctgcctttt caccctagct agaagctggg
       61 ataggatttg ctttgcacct gccttttcac ctagctagaa gctgggatag gatttgcttt
      121 gcacctgcct tttcacccta gctagaagct gggataggat ttgctttgca cctgcctttt
      181 cacctagcta gaagctggga taggatttgc tttgcacctg ccttttcacc ctagctagaa
      241 gctgggatag gatttgcttt gcacctgcct tttcacctag ctagaagctg ggataggatt
      301 tgctttgcac ctgccttttc accctagggg taccgatcga tagatctatt taaattaaac
      361 tgggcatcag cgcaattcaa ttggtttggt aatagcaagg gaaaatagaa tgaagtgatc
      421 tccaaaaaat aagtactttt tgactgtaaa taaaattgta aggagtaaaa agtacttttt
      481 tttctaaaaa aatgtaatta agtaaaagta aaagtattga tttttaattg tactcaagta
      541 aagtaaaaat ccccaaaaat aatacttaag tacagtaatc aagtaaaatt actcaagtac
      601 tttacacctc tgggcccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg
      661 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
      721 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
      781 agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg
      841 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
      901 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
      961 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     1021 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     1081 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     1141 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     1201 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     1261 ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catcaggtgg
     1321 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1381 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     1441 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     1501 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     1561 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     1621 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     1681 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     1741 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     1801 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     1861 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     1921 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     1981 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     2041 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     2101 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2161 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     2221 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     2281 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     2341 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     2401 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2461 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2521 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2581 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     2641 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2701 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2761 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2821 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2881 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2941 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3001 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3061 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3121 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3181 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3241 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     3301 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     3361 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     3421 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     3481 gctatttagg tgacactata gaatactcaa gctatgcatc caacgcgttg ggagctctcc
     3541 catatggtcg agcagaggtg taaaaagtac tcaaaaattt tactcaagtg aaagtacaag
     3601 tacttaggga aaattttact caattaaaag taaaagtatc tggctagaat cttacttgag
     3661 taaaagtaaa aaagtactcc attaaaattg tacttgagta ttaaggaagt aaaagtaaaa
     3721 gcaagaaaga aaactagaga ttcttgttta agcttaatta aaaaacctcc cacacctccc
     3781 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     3841 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3901 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctacgt
     3961 aatacgactc actatagttc tagtcaggca ccgggcttgc gggtcatgca ccaggtgcgc
     4021 ggtccttcgg gcacctcgac gtcggcggtg acggtgaagc cgagccgctc gtagaagggg
     4081 aggttgcggg gcgcggaggt ctccaggaag gcgggcaccc cggcgcgctc ggccgcctcc
     4141 actccgggga gcacgacggc gctgcccaga cccttgccct ggtggtcggg cgagacgccg
     4201 acggtggcca ggaaccacgc gggctccttg ggccggtgcg gcgccaggag gccttccatc
     4261 tgttgctgcg cggccagccg ggaaccgctc aactcggcca tgcgcgggcc gatctcggcg
     4321 aacaccgccc ccgcttcgac gctctccggc gtggtccaga ccgccaccgc ggcgccgtcg
     4381 tccgcgaccc acaccttgcc gatgtcgagc ccgacgcgcg tgaggaagag ttcttgcagc
     4441 tcggtgaccc gctcgatgtg gcggtccgga tcgacggtgt ggcgcgtggc ggggtagtcg
     4501 gcgaacgcgg cggcgagggt gcgtacggcc ctggggacgt cgtcgcgggt ggcgaggcgc
     4561 accgtgggct tgtactcggt catggtggct tcgagactag tgaaatcgtc gacggtttag
     4621 ttcctcacct tgtcgtatta tactatgccg atatactatg ccgatgatta attgtcaaca
     4681 ggctgcaggt cgaaaggccc ggagatgagg aagaggagaa cagcgcggca gacgtgcgct
     4741 tttgaagcgt gcagaatgcc gggcctccgg aggaccttcg ggcgcccgcc ccgcccctga
     4801 gcccgcccct gagcccgccc ccggacccac cccttcccag cctctgagcc cagaaagcga
     4861 aggagcaaag ctgctattgg ccgctgcccc aaaggcctac ccgcttccat tgctcagcgg
     4921 tgctgtccat ctgcacgaga ctagtgagac gtgctacttc catttgtcac gtcctgcacg
     4981 acgcgagctg cggggcgggg gggaacttcc tgactagggg aggagtagaa ggtggcgcga
     5041 aggggccacc aaagaacgga gccggttggc gcctaccggt ggatgtggaa tgtgtgcgag
     5101 gccagaggcc acttgtgtag cgccaagtgc ccagcggggc tgctaaagcg catgctccag
     5161 actgccttgg gaaaagcgcc tcccctaccc ggtgcggccg cgaattaaaa aacctcccac
     5221 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     5281 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5341 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     5401 tctacgtaat acgactcact atagttctag actaatcgat gacctttctt ttcttttttg
     5461 gcctggagcc gctgccggtg ctgccggtgc catgccccag gaacaggtgg tggcggccct
     5521 cggagcgctc gtactgttcc acgatggtgt agtcctcgtt gtgggaggtg atgtccagct
     5581 tggtgtccac gtagtagtag ccgggcagtt gcacgggctt cttggccatg tagatggtct
     5641 tgaactccac caggtagtgg ccgccgtcct tcagcttcag ggcctggtgg atctcgccct
     5701 tcagcacgcc gtcgcggggg tacaggcgct cggtggaggc ctcccagccc atggtcttct
     5761 tctgcattac ggggccgtcg ggggggaagt tggtgccgcg catcttcacc ttgtagatca
     5821 gcgtgccgtc ctgcagggag gagtcctggg tcacggtcac cagaccgccg tcctcgaagt
     5881 tcatcacgcg ctcccacttg aagccctcgg ggaaggacag cttcttgtaa tcggggatgt
     5941 cggcggggtg cttcacgtac gccttggagc cgtacatgaa ctggggggac aggatgtccc
     6001 aggcgaaggg cagggggccg cccttggtca ccttcagctt ggcggtctgg gtgccctcgt
     6061 aggggcggcc ctcgccctcg ccctcgatct cgaactcgtg gccgttcatg gagccctcca
     6121 tgcgcacctt gaagcgcatg aactctttga tgacctcctc gcccttgctc accatggtcg
     6181 cggcctgagc ggtgtggagc agcctctctg cttgcacgct cgttttttaa agcgctccag
     6241 cccgaattgc gcgcccagtg acgtcagcgc agcctggctt cccaggcacc ccccgcccgg
     6301 ccccactttc cctccacttg tcccctccgg atccggcgcg ccgctagcta gaagctggga
     6361 taggatttgc tttgcacctg ccttttcacc ta
//
