LOCUS       sequence-312929-        6347 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 6347)
  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 6347)
  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. PGK-puro
FEATURES             Location/Qualifiers
     source          1..6347
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(397..584,585..596)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     promoter        complement(609..627)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(634..651)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(634..650)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(643..665)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      791..1246
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(878..897)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1088..1109
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1324..1428
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1429..1497,1498..2289)
                     /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(1647..1666)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2460..3048
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2949..2968
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3202..3219
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3336..3357
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3372..3377,3378..3395,3396..3402)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3410..3426
                     /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     3415..3437
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3434..3450
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3434..3450
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3468..3486
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3468..3485
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    join(3537..3548,3549..3711)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    3805..3939
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(3968..4567)
                     /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(4051..4071)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4548..4567
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4616..4663)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(4675..5174)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4723..4742)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     5116..5139
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     polyA_signal    5252..5386
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5419..6114)
                     /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(5694..5713)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5963..5981
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
ORIGIN
        1 ggatttgctt tgcacctgcc ttttcaccct agctagaagc tgggatagga tttgctttgc
       61 acctgccttt tcacctagct agaagctggg ataggatttg ctttgcacct gccttttcac
      121 cctagctaga agctgggata ggatttgctt tgcacctgcc ttttcaccta gctagaagct
      181 gggataggat ttgctttgca cctgcctttt caccctagct agaagctggg ataggatttg
      241 ctttgcacct gccttttcac ctagctagaa gctgggatag gatttgcttt gcacctgcct
      301 tttcacccta ggggtaccga tcgatagatc tatttaaatt aaactgggca tcagcgcaat
      361 tcaattggtt tggtaatagc aagggaaaat agaatgaagt gatctccaaa aaataagtac
      421 tttttgactg taaataaaat tgtaaggagt aaaaagtact tttttttcta aaaaaatgta
      481 attaagtaaa agtaaaagta ttgattttta attgtactca agtaaagtaa aaatccccaa
      541 aaataatact taagtacagt aatcaagtaa aattactcaa gtactttaca cctctgggcc
      601 caattcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga
      661 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
      721 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
      781 tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
      841 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
      901 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
      961 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     1021 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     1081 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     1141 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     1201 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt
     1261 ttctccttac gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat
     1321 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1381 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     1441 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     1501 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     1561 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     1621 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     1681 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     1741 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     1801 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     1861 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     1921 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     1981 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     2041 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     2101 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     2161 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     2221 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     2281 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     2341 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     2401 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2461 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2521 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2581 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     2641 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2701 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2761 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2821 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2881 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2941 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3001 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3061 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3121 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3181 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     3241 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     3301 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     3361 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     3421 taacaatttc acacaggaaa cagctatgac catgattacg ccaagctatt taggtgacac
     3481 tatagaatac tcaagctatg catccaacgc gttgggagct ctcccatatg gtcgagcaga
     3541 ggtgtaaaaa gtactcaaaa attttactca agtgaaagta caagtactta gggaaaattt
     3601 tactcaatta aaagtaaaag tatctggcta gaatcttact tgagtaaaag taaaaaagta
     3661 ctccattaaa attgtacttg agtattaagg aagtaaaagt aaaagcaaga aagaaaacta
     3721 gagattcttg tttaagctta attaaaaaac ctcccacacc tccccctgaa cctgaaacat
     3781 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     3841 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3901 ttgtccaaac tcatcaatgt atcttatcat gtctggatct acgtaatacg actcactata
     3961 gttctagtca ggcaccgggc ttgcgggtca tgcaccaggt gcgcggtcct tcgggcacct
     4021 cgacgtcggc ggtgacggtg aagccgagcc gctcgtagaa ggggaggttg cggggcgcgg
     4081 aggtctccag gaaggcgggc accccggcgc gctcggccgc ctccactccg gggagcacga
     4141 cggcgctgcc cagacccttg ccctggtggt cgggcgagac gccgacggtg gccaggaacc
     4201 acgcgggctc cttgggccgg tgcggcgcca ggaggccttc catctgttgc tgcgcggcca
     4261 gccgggaacc gctcaactcg gccatgcgcg ggccgatctc ggcgaacacc gcccccgctt
     4321 cgacgctctc cggcgtggtc cagaccgcca ccgcggcgcc gtcgtccgcg acccacacct
     4381 tgccgatgtc gagcccgacg cgcgtgagga agagttcttg cagctcggtg acccgctcga
     4441 tgtggcggtc cggatcgacg gtgtggcgcg tggcggggta gtcggcgaac gcggcggcga
     4501 gggtgcgtac ggccctgggg acgtcgtcgc gggtggcgag gcgcaccgtg ggcttgtact
     4561 cggtcatggt ggcttcgaga ctagtgaaat cgtcgacggt ttagttcctc accttgtcgt
     4621 attatactat gccgatatac tatgccgatg attaattgtc aacaggctgc aggtcgaaag
     4681 gcccggagat gaggaagagg agaacagcgc ggcagacgtg cgcttttgaa gcgtgcagaa
     4741 tgccgggcct ccggaggacc ttcgggcgcc cgccccgccc ctgagcccgc ccctgagccc
     4801 gcccccggac ccaccccttc ccagcctctg agcccagaaa gcgaaggagc aaagctgcta
     4861 ttggccgctg ccccaaaggc ctacccgctt ccattgctca gcggtgctgt ccatctgcac
     4921 gagactagtg agacgtgcta cttccatttg tcacgtcctg cacgacgcga gctgcggggc
     4981 gggggggaac ttcctgacta ggggaggagt agaaggtggc gcgaaggggc caccaaagaa
     5041 cggagccggt tggcgcctac cggtggatgt ggaatgtgtg cgaggccaga ggccacttgt
     5101 gtagcgccaa gtgcccagcg gggctgctaa agcgcatgct ccagactgcc ttgggaaaag
     5161 cgcctcccct acccggtgcg gccgcgaatt aaaaaacctc ccacacctcc ccctgaacct
     5221 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     5281 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     5341 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatctacg taatacgact
     5401 cactatagtt ctagattact tgtacagctc gtccatgccg ccggtggagt ggcggccctc
     5461 ggagcgttcg tactgttcca ccacggtgta gtcctcgttg tgggaggtga tgtccaactt
     5521 gcggtccacg ttgtaggcgc cgggcatctg cacgggcttc ttggccttgt aggtggtctt
     5581 gaagtccgcc aggtagcgtc cgccgtcctt caggcgcagg gccatcttaa tgtcgccctt
     5641 cagcacgccg tcctcggggt acaaccgctc ggtggacgct tcccagccca ttgtcttctt
     5701 ctgcattacg gggccgtcag gagggaagtt ggtgccgcgg agcttcacct tgtagatcag
     5761 ggtgccgtcc tccagggagg tgtcctgggt cacggtcacg gcgccgccgt cctcgaagtt
     5821 catcacgcgc tcccacttga agccctcggg gaaggactgc ttatagtagt cggggatgtc
     5881 ggcggggtgc ttgatgaagg ccctggagcc gtacatgaac tgaggggaca ggatgtccca
     5941 ggagaagggc agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta
     6001 ggggcggccc tcgccctcgc cctcgatctc gaactcgtgg ccgttcatgg agccctccat
     6061 gtgcaccttg aaccgcatga actccttgat cactgcctcg cccttgctca ccatggtcgc
     6121 ggcctgagcg gtgtggagca gcctctctgc ttgcacgctc gttttttaaa gcgctccagc
     6181 ccgaattgcg cgcccagtga cgtcagcgca gcctggcttc ccaggcaccc cccgcccggc
     6241 cccactttcc ctccacttgt cccctccgga tccggcgcgc cgctagctag aagctgggat
     6301 aggatttgct ttgcacctgc cttttcacct agctagaagc tgggata
//
