LOCUS       sequence-312926-        6359 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 6359)
  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 6359)
  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. PGK-puro
FEATURES             Location/Qualifiers
     source          1..6359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(626..644)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     894..913
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1088..1111
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1233..1367
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        1445..1944
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1480..1503)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1877..1896
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        1956..2003
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2052..2651
                     /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(2052..2071)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2548..2568
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2680..2814
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(2908..3070,3071..3082)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     promoter        complement(3133..3151)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3134..3151)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3169..3185)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3169..3185)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3182..3204)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3193..3209
                     /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(3217..3223,3224..3241,3242..3247))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3262..3283
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3400..3417)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3571..4159)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3651..3670)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4330..5121,5122..5190))
                     /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     4953..4972
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5191..5295)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5373..5828)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5510..5531)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5722..5741
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5954..5976
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5968..5985
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5969..5985
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5992..6010
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(6023..6034,6035..6222)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 tgaaaaggca ggtgcaaagc aaatcctatc ccagcttcta gctagggtga aaaggcaggt
       61 gcaaagcaaa tcctatccca gcttctagct aggtgaaaag gcaggtgcaa agcaaatcct
      121 atcccagctt ctagctaggg tgaaaaggca ggtgcaaagc aaatcctatc ccagcttcta
      181 gctaggtgaa aaggcaggtg caaagcaaat cctatcccag cttctagcta gggtgaaaag
      241 gcaggtgcaa agcaaatcct atcccagctt ctagctaggt gaaaaggcag gtgcaaagca
      301 aatcctatcc cagcttctag ctagcggcgc gccggatccg gaggggacaa gtggagggaa
      361 agtggggccg ggcggggggt gcctgggaag ccaggctgcg ctgacgtcac tgggcgcgca
      421 attcgggctg gagcgcttta aaaaacgagc gtgcaagcag agaggctgct ccacaccgct
      481 caggccgcga ccatggtgag caagggcgag gaggtcatca aagagttcat gcgcttcaag
      541 gtgcgcatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc
      601 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc
      661 ttcgcctggg acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac
      721 cccgccgaca tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc
      781 gtgatgaact tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac
      841 ggcacgctga tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta
      901 atgcagaaga agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc
      961 gtgctgaagg gcgagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg
     1021 gagttcaaga ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg
     1081 gacaccaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag
     1141 cgctccgagg gccgccacca cctgttcctg gggcatggca ccggcagcac cggcagcggc
     1201 taatctagaa ctatagtgag tcgtattacg tagatccaga catgataaga tacattgatg
     1261 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     1321 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     1381 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa ttcgcggccg
     1441 caccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg
     1501 ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt
     1561 aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta
     1621 gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta
     1681 gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc
     1741 ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga
     1801 aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg
     1861 tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc
     1921 tcttcctcat ctccgggcct ttcgacctgc agcctgttga caattaatca tcggcatagt
     1981 atatcggcat agtataatac gacaaggtga ggaactaaac cgtcgacgat ttcactagtc
     2041 tcgaagccac catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     2101 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     2161 tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     2221 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     2281 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     2341 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     2401 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca
     2461 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     2521 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     2581 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     2641 ccggtgcctg actagaacta tagtgagtcg tattacgtag atccagacat gataagatac
     2701 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     2761 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     2821 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaatta
     2881 agcttaaaca agaatctcta gttttctttc ttgcttttac ttttacttcc ttaatactca
     2941 agtacaattt taatggagta cttttttact tttactcaag taagattcta gccagatact
     3001 tttactttta attgagtaaa attttcccta agtacttgta ctttcacttg agtaaaattt
     3061 ttgagtactt tttacacctc tgctcgacca tatgggagag ctcccaacgc gttggatgca
     3121 tagcttgagt attctatagt gtcacctaaa tagcttggcg taatcatggt catagctgtt
     3181 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     3241 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     3301 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3361 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3421 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3481 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3541 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3601 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3661 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3721 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3781 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3841 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3901 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3961 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4021 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4081 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4141 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4201 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4261 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4321 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4381 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4441 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     4501 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4561 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4621 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4681 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4741 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4801 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4861 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4921 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4981 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5041 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5101 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5161 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5221 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5281 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt gaaataccgc
     5341 acagatgcgt aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa
     5401 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     5461 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     5521 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     5581 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     5641 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     5701 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     5761 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     5821 gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     5881 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     5941 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac
     6001 tcactatagg gcgaattggg cccagaggtg taaagtactt gagtaatttt acttgattac
     6061 tgtacttaag tattattttt ggggattttt actttacttg agtacaatta aaaatcaata
     6121 cttttacttt tacttaatta cattttttta gaaaaaaaag tactttttac tccttacaat
     6181 tttatttaca gtcaaaaagt acttattttt tggagatcac ttcattctat tttcccttgc
     6241 tattaccaaa ccaattgaat tgcgctgatg cccagtttaa tttaaataga tctatcgatc
     6301 ggtaccccta gggtgaaaag gcaggtgcaa agcaaatcct atcccagctt ctagctagg
//
