LOCUS       sequence-312920-        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-mNeonGreen-NLS. PGK-puro
FEATURES             Location/Qualifiers
     source          1..6392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             552..1259
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      807..816
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1266..1286
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1325..1459
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        1537..2036
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1572..1595)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1969..1988
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2048..2095
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2144..2743
                     /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(2144..2163)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2640..2660
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2772..2906
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(3000..3162,3163..3174)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     promoter        complement(3225..3243)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3226..3243)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3261..3277)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3261..3277)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3274..3296)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3285..3301
                     /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(3309..3315,3316..3333,3334..3339))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3354..3375
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3492..3509)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3663..4251)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3743..3762)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4422..5213,5214..5282))
                     /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     5045..5064
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5283..5387)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5465..5920)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5602..5623)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5814..5833
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6046..6068
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6060..6077
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6061..6077
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6084..6102
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(6115..6126,6127..6314)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
ORIGIN
        1 ggtaccccta gggtgaaaag gcaggtgcaa agcaaatcct atcccagctt ctagctaggt
       61 gaaaaggcag gtgcaaagca aatcctatcc cagcttctag ctagggtgaa aaggcaggtg
      121 caaagcaaat cctatcccag cttctagcta ggtgaaaagg caggtgcaaa gcaaatccta
      181 tcccagcttc tagctagggt gaaaaggcag gtgcaaagca aatcctatcc cagcttctag
      241 ctaggtgaaa aggcaggtgc aaagcaaatc ctatcccagc ttctagctag ggtgaaaagg
      301 caggtgcaaa gcaaatccta tcccagcttc tagctaggtg aaaaggcagg tgcaaagcaa
      361 atcctatccc agcttctagc tagcggcgcg ccggatccgg aggggacaag tggagggaaa
      421 gtggggccgg gcggggggtg cctgggaagc caggctgcgc tgacgtcact gggcgcgcaa
      481 ttcgggctgg agcgctttaa aaaacgagcg tgcaagcaga gaggctgctc cacaccgctc
      541 aggccgcgac catggtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
      601 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
      661 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
      721 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
      781 accctgacgg gatgtcgcct ttccaggccg ccatggtaga tggctccgga taccaagtcc
      841 atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
      901 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
      961 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
     1021 acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat ggcaagcgct
     1081 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
     1141 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
     1201 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaagt
     1261 ccaggccaaa aaagaaaaga aaggtcatcg attagtctag aactatagtg agtcgtatta
     1321 cgtagatcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     1381 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     1441 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     1501 ggaggtgtgg gaggtttttt aattcgcggc cgcaccgggt aggggaggcg cttttcccaa
     1561 ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc
     1621 tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg
     1681 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc
     1741 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg
     1801 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt
     1861 gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg
     1921 gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc
     1981 ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc ctttcgacct
     2041 gcagcctgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt
     2101 gaggaactaa accgtcgacg atttcactag tctcgaagcc accatgaccg agtacaagcc
     2161 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     2221 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     2281 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     2341 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     2401 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     2461 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     2521 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     2581 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     2641 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     2701 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgactagaac tatagtgagt
     2761 cgtattacgt agatccagac atgataagat acattgatga gtttggacaa accacaacta
     2821 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     2881 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     2941 ttcaggggga ggtgtgggag gttttttaat taagcttaaa caagaatctc tagttttctt
     3001 tcttgctttt acttttactt ccttaatact caagtacaat tttaatggag tactttttta
     3061 cttttactca agtaagattc tagccagata cttttacttt taattgagta aaattttccc
     3121 taagtacttg tactttcact tgagtaaaat ttttgagtac tttttacacc tctgctcgac
     3181 catatgggag agctcccaac gcgttggatg catagcttga gtattctata gtgtcaccta
     3241 aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3301 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     3361 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3421 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3481 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3541 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3601 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3661 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3721 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3781 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3841 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3901 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3961 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4021 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4081 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4141 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4201 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4261 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4321 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4381 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4441 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4501 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4561 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4621 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4681 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4741 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4801 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4861 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4921 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4981 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5041 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5101 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5161 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5221 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5281 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5341 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5401 aaagtgccac ctgatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     5461 caggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5521 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5581 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5641 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5701 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5761 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5821 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5881 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc
     5941 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     6001 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     6061 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggcccagagg
     6121 tgtaaagtac ttgagtaatt ttacttgatt actgtactta agtattattt ttggggattt
     6181 ttactttact tgagtacaat taaaaatcaa tacttttact tttacttaat tacatttttt
     6241 tagaaaaaaa agtacttttt actccttaca attttattta cagtcaaaaa gtacttattt
     6301 tttggagatc acttcattct attttccctt gctattacca aaccaattga attgcgctga
     6361 tgcccagttt aatttaaata gatctatcga tc
//
