LOCUS       sequence-312894-        6359 bp    DNA     circular SYN 14-APR-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   Oost KC, van Voorthuijsen L, Fumagalli A, Lindeboom RGH, Sprangers 
            J, Omerzu M, Rodriguez-Colman MJ, Heinz MC, Verlaan-Klink I, Maurice
            MM, Burgering BMT, van Rheenen J, Vermeulen M, Snippert HJG
  TITLE     Specific Labeling of Stem Cell Activity in Human Colorectal 
            Organoids Using an ASCL2-Responsive Minigene.
  JOURNAL   Cell Rep. 2018 Feb 6;22(6):1600-1614. doi: 
            10.1016/j.celrep.2018.01.033.
  PUBMED    29425513
REFERENCE   2  (bases 1 to 6359)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2018.01"; journalName: "Cell Rep"; date: 
            "2018-02-6- 6"; volume: "22"; issue: "6"; pages: "1600-1614"
COMMENT     Sequence Label: Tol2 8xSTAR-mNeonGreen. PGK-puro
FEATURES             Location/Qualifiers
     source          1..6359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             830..1540
                     /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      1085..1094
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1570..1704
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        1782..2281
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1817..1840)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2214..2233
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2293..2340
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2389..2988
                     /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(2389..2408)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2885..2905
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3017..3151
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(3470..3488)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3471..3488)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3506..3522)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3506..3522)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3519..3541)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3530..3546
                     /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(3554..3560,3561..3578,3579..3584))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3599..3620
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3737..3754)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3908..4496)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3988..4007)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4667..5458,5459..5527))
                     /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     5290..5309
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5528..5632)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5710..6165)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5847..5868)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6059..6078
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6291..6313
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6305..6322
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6306..6322
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6329..6347
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cagaggtgta aagtacttga gtaattttac ttgattactg tacttaagta ttatttttgg
       61 ggatttttac tttacttgag tacaattaaa aatcaatact tttactttta cttaattaca
      121 tttttttaga aaaaaaagta ctttttactc cttacaattt tatttacagt caaaaagtac
      181 ttattttttg gagatcactt cattctattt tcccttgcta ttaccaaacc aattgaattg
      241 cgctgatgcc cagtttaatt taaatagatc tatcgatcgg tacccctagg gtgaaaaggc
      301 aggtgcaaag caaatcctat cccagcttct agctaggtga aaaggcaggt gcaaagcaaa
      361 tcctatccca gcttctagct agggtgaaaa ggcaggtgca aagcaaatcc tatcccagct
      421 tctagctagg tgaaaaggca ggtgcaaagc aaatcctatc ccagcttcta gctagggtga
      481 aaaggcaggt gcaaagcaaa tcctatccca gcttctagct aggtgaaaag gcaggtgcaa
      541 agcaaatcct atcccagctt ctagctaggg tgaaaaggca ggtgcaaagc aaatcctatc
      601 ccagcttcta gctaggtgaa aaggcaggtg caaagcaaat cctatcccag cttctagcta
      661 gcggcgcgcc ggatccggag gggacaagtg gagggaaagt ggggccgggc ggggggtgcc
      721 tgggaagcca ggctgcgctg acgtcactgg gcgcgcaatt cgggctggag cgctttaaaa
      781 aacgagcgtg caagcagaga ggctgctcca caccgctcag gccgcgacca tggtgagcaa
      841 gggcgaggag gataacatgg cctctctccc agcgacacat gagttacaca tctttggctc
      901 catcaacggt gtggactttg acatggtggg tcagggcacc ggcaatccaa atgatggtta
      961 tgaggagtta aacctgaagt ccaccaaggg tgacctccag ttctccccct ggattctggt
     1021 ccctcatatc gggtatggct tccatcagta cctgccctac cctgacggga tgtcgccttt
     1081 ccaggccgcc atggtagatg gctccggata ccaagtccat cgcacaatgc agtttgaaga
     1141 tggtgcctcc cttactgtta actaccgcta cacctacgag ggaagccaca tcaaaggaga
     1201 ggcccaggtg aaggggactg gtttccctgc tgacggtcct gtgatgacca actcgctgac
     1261 cgctgcggac tggtgcaggt cgaagaagac ttaccccaac gacaaaacca tcatcagtac
     1321 ctttaagtgg agttacacca ctggaaatgg caagcgctac cggagcactg cgcggaccac
     1381 ctacaccttt gccaagccaa tggcggctaa ctatctgaag aaccagccga tgtacgtgtt
     1441 ccgtaagacg gagctcaagc actccaagac cgagctcaac ttcaaggagt ggcaaaaggc
     1501 ctttaccgat gtgatgggca tggacgagct gtacaagtaa tctagaacta tagtgagtcg
     1561 tattacgtag atccagacat gataagatac attgatgagt ttggacaaac cacaactaga
     1621 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     1681 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     1741 cagggggagg tgtgggaggt tttttaattc gcggccgcac cgggtagggg aggcgctttt
     1801 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag
     1861 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     1921 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc
     1981 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     2041 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     2101 gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct
     2161 caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg
     2221 cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc
     2281 gacctgcagc ctgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
     2341 aaggtgagga actaaaccgt cgacgatttc actagtctcg aagccaccat gaccgagtac
     2401 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     2461 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     2521 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     2581 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     2641 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     2701 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     2761 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     2821 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     2881 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     2941 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgact agaactatag
     3001 tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg gacaaaccac
     3061 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     3121 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     3181 tcaggttcag ggggaggtgt gggaggtttt ttaattaagc ttaaacaaga atctctagtt
     3241 ttctttcttg cttttacttt tacttcctta atactcaagt acaattttaa tggagtactt
     3301 ttttactttt actcaagtaa gattctagcc agatactttt acttttaatt gagtaaaatt
     3361 ttccctaagt acttgtactt tcacttgagt aaaatttttg agtacttttt acacctctgc
     3421 tcgaccatat gggagagctc ccaacgcgtt ggatgcatag cttgagtatt ctatagtgtc
     3481 acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3541 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3601 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3661 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3721 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3781 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3841 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3901 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3961 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4021 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4081 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4141 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4201 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4261 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4321 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4381 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4441 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4501 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4561 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4621 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4681 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4741 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4801 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4861 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4921 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4981 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5041 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5101 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5161 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5221 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5281 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5341 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5401 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5461 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5521 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5581 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5641 cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     5701 cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     5761 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     5821 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     5881 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     5941 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     6001 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     6061 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     6121 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag
     6181 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     6241 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     6301 acgttgtaaa acgacggcca gtgaattgta atacgactca ctatagggcg aattgggcc
//
