LOCUS       sequence-312890-        8384 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 8384)
  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 8384)
  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"; 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: pLV 4xSTAR-mTagBFP2-blast
FEATURES             Location/Qualifiers
     source          1..8384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    797..1349
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(941..958)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1168..1187
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      1370..1379
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1376..1774
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             2617..2850
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2922..3043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2959..2978)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3013..3032
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3083..3218
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3238..3257)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3239..3257)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3267..3284)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3267..3283)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3276..3298)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3425..3880
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3512..3531)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3722..3743
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3906..4010
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4011..4079,4080..4871)
                     /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(4229..4248)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5042..5630
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5531..5550
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5784..5801
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5918..5939
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5954..5959,5960..5977,5978..5984)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5992..6008
                     /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     5997..6019
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6016..6032
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6016..6032
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6051..6071
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6053..6071
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6099..6325
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6326..6506
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6553..6678
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7171..7404
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7589..7633
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    7895..8012
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 atggtgtcta agggcgaaga actgatcaaa gaaaatatgc atatgaaact gtatatggaa
       61 ggaacagtgg ataatcacca ttttaaatgt accagcgaag gagagggaaa accttatgaa
      121 ggaacacaga caatgcgaat taaagtcgtg gaaggagggc ccctcccttt tgcctttgat
      181 attctggcta cttcctttct ctatggatcc aagacattta ttaatcacac acagggaatt
      241 cctgattttt tcaaacagag ctttcctgaa gggtttacct gggaacgggt gacaacctat
      301 gaggatggcg gggtgctgac agctacacag gatacatccc tccaggatgg gtgtctcatt
      361 tataatgtca aaattcgggg cgtgaatttt accagcaatg gacccgtgat gcagaaaaag
      421 accctcggat gggaagcctt tacagaaacg ctgtatcctg ctgatggagg gctggagggg
      481 aggaacgata tggccctgaa actcgtggga ggcagccatc tgattgcaaa tgccaaaaca
      541 acctatagga gcaaaaagcc tgctaagaat ctcaaaatgc ctggagtcta ttatgtggat
      601 tacaggctgg agaggattaa agaagccaac aacgaaacat atgtcgaaca gcatgaagtg
      661 gcagtggcca ggtattgtga tctcccctcc aagctgggcc ataagctttc tagacagcgc
      721 tcaccggtcg ttaacattaa gctagcctag tgccatttgt tcagtggccc gggaattccg
      781 cccctctccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt
      841 gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct
      901 ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa
      961 ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg
     1021 tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc
     1081 caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg
     1141 agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg
     1201 aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc
     1261 tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg
     1321 gttttccttt gaaaaacacg atgataatat ggatatggcc acaacggtcg ccaccatggc
     1381 caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag
     1441 catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat
     1501 cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct
     1561 gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga
     1621 gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc
     1681 tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga
     1741 attgctgccc tctggttatg tgtgggaggg ctaaatgcat gtattcaatc taagcaggct
     1801 ttcactttct cgccaactta caaggccttt ctgtgtaaac aatacctgaa cctttacccc
     1861 gttgcccggc aacggccagg tctgtgccaa gtgtttgctg acgcaacccc cactggctgg
     1921 ggcttggtca tgggccatca gcgcatgcgt ggaacctttt cggctcctct gccgatccat
     1981 actgcggaac tcctagccgc ttgttttgct cgcagcaggt ctggagcaaa cattatcggg
     2041 actgataact ctgttgtcct atcccgcaaa tatacatcgt ttccatggct gctaggctgt
     2101 gctgccaact ggatcctgcg cgggacgtcc tttgtttacg tcccgtcggc gctgaatcct
     2161 gcggacgacc cttctcgggg tcgcttggga ctctctcgtc cccttctccg tctgccgttc
     2221 cgaccgacca cggggcgcac ctctctttac gcggactccc cgtctgtgcc ttctcatctg
     2281 ccggaccgtg tgcacttcgc ttcacctctg cacgtcgcat ggagaccacc gtgaacgccc
     2341 accaaatatt gcccaaggtc ttacataaga ggactcttgg actctcagca atgtcaacga
     2401 ccgaccttga ggcatacttc aaagactgtt tgtttaaaga ctgggaggag ttgggggagg
     2461 agattaggtt aaaggtcttt gtactaggag gctgtaggca taaattggtc tgcgcaccag
     2521 caccatggcg caatcactag agcggggtac ctttaagacc aatgacttac aaggcagctg
     2581 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
     2641 gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct
     2701 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     2761 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     2821 ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt
     2881 atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt
     2941 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3001 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc
     3061 tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3121 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     3181 gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc
     3241 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     3301 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     3361 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     3421 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3481 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3541 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3601 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3661 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3721 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     3781 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     3841 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     3901 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3961 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4021 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4081 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4141 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4201 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4261 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4321 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4381 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4441 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4501 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4561 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4621 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4681 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     4741 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4801 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4861 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4921 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4981 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5041 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5101 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5161 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5221 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5281 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5341 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5401 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5461 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5521 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5581 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5641 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5701 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5761 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     5821 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     5881 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     5941 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     6001 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
     6061 ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg caatactctt
     6121 gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag agaaaaagca
     6181 ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt gccttattag gaaggcaaca
     6241 gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag atattgtatt
     6301 taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct gagcctggga
     6361 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     6421 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     6481 ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg
     6541 aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc
     6601 gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga
     6661 gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt
     6721 cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg
     6781 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa
     6841 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat
     6901 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa
     6961 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc
     7021 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
     7081 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
     7141 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
     7201 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
     7261 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
     7321 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
     7381 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
     7441 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
     7501 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
     7561 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
     7621 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
     7681 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
     7741 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
     7801 gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc
     7861 cattcgatta gtgaacggat ctcgacggta tcggttttaa aagaaaaggg gggattgggg
     7921 ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat
     7981 tacaaaaaca aattacaaaa attcaaaatt ttatcgatcg gtacccctag ggtgaaaagg
     8041 caggtgcaaa gcaaatccta tcccagcttc tagctaggtg aaaaggcagg tgcaaagcaa
     8101 atcctatccc agcttctagc tagggtgaaa aggcaggtgc aaagcaaatc ctatcccagc
     8161 ttctagctag gtgaaaaggc aggtgcaaag caaatcctat cccagcttct agctagcggc
     8221 gcgccggatc cggaggggac aagtggaggg aaagtggggc cgggcggggg gtgcctggga
     8281 agccaggctg cgctgacgtc actgggcgcg caattcgggc tggagcgctt taaaaaacga
     8341 gcgtgcaagc agagaggctg ctccacaccg ctcaggccgc gacc
//
