LOCUS       sequence-312924-        6380 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 6380)
  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 6380)
  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..6380
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(378..565,566..577)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     promoter        complement(590..608)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(615..632)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(615..631)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(624..646)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      772..1227
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(859..878)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1069..1090
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1305..1409
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1410..1478,1479..2270)
                     /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(1628..1647)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2441..3029
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2930..2949
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3183..3200
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3317..3338
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3353..3358,3359..3376,3377..3383)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3391..3407
                     /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     3396..3418
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3415..3431
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3415..3431
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3449..3467
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3449..3466
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    join(3518..3529,3530..3692)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    3786..3920
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(3949..4548)
                     /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(4032..4052)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4529..4548
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4597..4644)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(4656..5155)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4704..4723)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     5097..5120
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     polyA_signal    5233..5367
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5406..5426)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5433..6128)
                     /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(5708..5727)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5977..5995
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
ORIGIN
        1 cttttcaccc tagctagaag ctgggatagg atttgctttg cacctgcctt ttcacctagc
       61 tagaagctgg gataggattt gctttgcacc tgccttttca ccctagctag aagctgggat
      121 aggatttgct ttgcacctgc cttttcacct agctagaagc tgggatagga tttgctttgc
      181 acctgccttt tcaccctagc tagaagctgg gataggattt gctttgcacc tgccttttca
      241 cctagctaga agctgggata ggatttgctt tgcacctgcc ttttcaccct aggggtaccg
      301 atcgatagat ctatttaaat taaactgggc atcagcgcaa ttcaattggt ttggtaatag
      361 caagggaaaa tagaatgaag tgatctccaa aaaataagta ctttttgact gtaaataaaa
      421 ttgtaaggag taaaaagtac ttttttttct aaaaaaatgt aattaagtaa aagtaaaagt
      481 attgattttt aattgtactc aagtaaagta aaaatcccca aaaataatac ttaagtacag
      541 taatcaagta aaattactca agtactttac acctctgggc ccaattcgcc ctatagtgag
      601 tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
      661 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
      721 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gacgcgccct
      781 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      841 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      901 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
      961 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1021 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1081 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1141 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1201 ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg
     1261 cggtatttca caccgcatca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1321 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1381 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1441 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1501 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1561 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1621 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1681 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1741 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1801 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1861 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1921 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     1981 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2041 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2101 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2161 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2221 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2281 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2341 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2401 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2461 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2521 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2581 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2641 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2701 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2761 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2821 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2881 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2941 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3001 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3061 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3121 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3181 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     3241 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     3301 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     3361 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     3421 acagctatga ccatgattac gccaagctat ttaggtgaca ctatagaata ctcaagctat
     3481 gcatccaacg cgttgggagc tctcccatat ggtcgagcag aggtgtaaaa agtactcaaa
     3541 aattttactc aagtgaaagt acaagtactt agggaaaatt ttactcaatt aaaagtaaaa
     3601 gtatctggct agaatcttac ttgagtaaaa gtaaaaaagt actccattaa aattgtactt
     3661 gagtattaag gaagtaaaag taaaagcaag aaagaaaact agagattctt gtttaagctt
     3721 aattaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     3781 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3841 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3901 tatcttatca tgtctggatc tacgtaatac gactcactat agttctagtc aggcaccggg
     3961 cttgcgggtc atgcaccagg tgcgcggtcc ttcgggcacc tcgacgtcgg cggtgacggt
     4021 gaagccgagc cgctcgtaga aggggaggtt gcggggcgcg gaggtctcca ggaaggcggg
     4081 caccccggcg cgctcggccg cctccactcc ggggagcacg acggcgctgc ccagaccctt
     4141 gccctggtgg tcgggcgaga cgccgacggt ggccaggaac cacgcgggct ccttgggccg
     4201 gtgcggcgcc aggaggcctt ccatctgttg ctgcgcggcc agccgggaac cgctcaactc
     4261 ggccatgcgc gggccgatct cggcgaacac cgcccccgct tcgacgctct ccggcgtggt
     4321 ccagaccgcc accgcggcgc cgtcgtccgc gacccacacc ttgccgatgt cgagcccgac
     4381 gcgcgtgagg aagagttctt gcagctcggt gacccgctcg atgtggcggt ccggatcgac
     4441 ggtgtggcgc gtggcggggt agtcggcgaa cgcggcggcg agggtgcgta cggccctggg
     4501 gacgtcgtcg cgggtggcga ggcgcaccgt gggcttgtac tcggtcatgg tggcttcgag
     4561 actagtgaaa tcgtcgacgg tttagttcct caccttgtcg tattatacta tgccgatata
     4621 ctatgccgat gattaattgt caacaggctg caggtcgaaa ggcccggaga tgaggaagag
     4681 gagaacagcg cggcagacgt gcgcttttga agcgtgcaga atgccgggcc tccggaggac
     4741 cttcgggcgc ccgccccgcc cctgagcccg cccctgagcc cgcccccgga cccacccctt
     4801 cccagcctct gagcccagaa agcgaaggag caaagctgct attggccgct gccccaaagg
     4861 cctacccgct tccattgctc agcggtgctg tccatctgca cgagactagt gagacgtgct
     4921 acttccattt gtcacgtcct gcacgacgcg agctgcgggg cgggggggaa cttcctgact
     4981 aggggaggag tagaaggtgg cgcgaagggg ccaccaaaga acggagccgg ttggcgccta
     5041 ccggtggatg tggaatgtgt gcgaggccag aggccacttg tgtagcgcca agtgcccagc
     5101 ggggctgcta aagcgcatgc tccagactgc cttgggaaaa gcgcctcccc tacccggtgc
     5161 ggccgcgaat taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     5221 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5281 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     5341 atcaatgtat cttatcatgt ctggatctac gtaatacgac tcactatagt tctagactaa
     5401 tcgatgacct ttcttttctt ttttggcctg gacttgtaca gctcgtccat gccgccggtg
     5461 gagtggcggc cctcggagcg ttcgtactgt tccaccacgg tgtagtcctc gttgtgggag
     5521 gtgatgtcca acttgcggtc cacgttgtag gcgccgggca tctgcacggg cttcttggcc
     5581 ttgtaggtgg tcttgaagtc cgccaggtag cgtccgccgt ccttcaggcg cagggccatc
     5641 ttaatgtcgc ccttcagcac gccgtcctcg gggtacaacc gctcggtgga cgcttcccag
     5701 cccattgtct tcttctgcat tacggggccg tcaggaggga agttggtgcc gcggagcttc
     5761 accttgtaga tcagggtgcc gtcctccagg gaggtgtcct gggtcacggt cacggcgccg
     5821 ccgtcctcga agttcatcac gcgctcccac ttgaagccct cggggaagga ctgcttatag
     5881 tagtcgggga tgtcggcggg gtgcttgatg aaggccctgg agccgtacat gaactgaggg
     5941 gacaggatgt cccaggagaa gggcaggggg ccacccttgg tcaccttcag cttggcggtc
     6001 tgggtgccct cgtaggggcg gccctcgccc tcgccctcga tctcgaactc gtggccgttc
     6061 atggagccct ccatgtgcac cttgaaccgc atgaactcct tgatcactgc ctcgcccttg
     6121 ctcaccatgg tcgcggcctg agcggtgtgg agcagcctct ctgcttgcac gctcgttttt
     6181 taaagcgctc cagcccgaat tgcgcgccca gtgacgtcag cgcagcctgg cttcccaggc
     6241 accccccgcc cggccccact ttccctccac ttgtcccctc cggatccggc gcgccgctag
     6301 ctagaagctg ggataggatt tgctttgcac ctgccttttc acctagctag aagctgggat
     6361 aggatttgct ttgcacctgc
//
