LOCUS       sequence-482793-        8241 bp    DNA     circular SYN 11-AUG-2025
DEFINITION  The p27 reporter vector, which expresses mVenus-fused, mutant (K–) 
            p27 protein with a defective CDK binding..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8241)
  AUTHORS   Ohta Y, Fujii M, Takahashi S, Takano A, Nanki K, Matano M, Hanyu H, 
            Saito M, Shimokawa M, Nishikori S, Hatano Y, Ishii R, Sawada K, 
            Machinaga A, Ikeda W, Imamura T, Sato T
  TITLE     Cell-matrix interface regulates dormancy in human colon cancer stem 
            cells.
  JOURNAL   Nature. 2022 Jul 7. pii: 10.1038/s41586-022-05043-y. doi: 
            10.1038/s41586-022-05043-y.
  PUBMED    35798028
REFERENCE   2  (bases 1 to 8241)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 11, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2022 Jul 7. pii: 10.1038/s41586-022-05043-y. doi: 
            10.1038/s41586-022-05043-y."
COMMENT     Sequence Label: p27-mVenus reporter (PB)
FEATURES             Location/Qualifiers
     source          1..8241
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(24..128)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(154..609)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(291..312)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     503..522
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     736..758
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     750..767
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     751..767
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   897..959
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1412..1623
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1636..1904
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      1931..1940
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1937..1939,1940..1942,1943..2653)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1982..2003)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2243..2262)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2590..2611
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(3319..3893,3894..3896,3897..3905)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3486..3503)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3713..3732
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3918..4712
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLAATGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3972..3991)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4582..4601
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4772..4792)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4834..4955
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4871..4890)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4925..4944
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5718..5752
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6243..6259)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6243..6259)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6256..6278)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6267..6283
                     /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(6291..6297,6298..6315,6316..6321))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6336..6357
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6474..6491)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6645..7233)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6725..6744)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7404..8195,8196..8241,1..23))
                     /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     8027..8046
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
       61 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
      121 gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa
      181 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
      241 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
      301 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
      361 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
      421 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
      481 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
      541 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
      601 agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
      661 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
      721 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcctcgt
      781 tcattcacgt ttttgaaccc gtggaggacg ggcagactcg cggtgcaaat gtgttttaca
      841 gcgtgatgga gcagatgaag atgctcgaca cgctgcagaa cacgcagcta gattaaccct
      901 agaaagataa tcatattgtg acgtacgtta aagataatca tgtgtaaaat tgacgcatgt
      961 gttttatcgg tctgtatatc gaggtttatt tattaatttg aatagatatt aagttttatt
     1021 atatttacac ttacatacta ataataaatt caacaaacaa tttatttatg tttatttatt
     1081 tattaaaaaa aacaaaaact caaaatttct tctataaagt aacaaaactt ttatgaggga
     1141 cagccccccc ccaaagcccc cagggatgta attacgtccc tcccccgcta gggggcagca
     1201 gcgagccgcc cggggctccg ctccggtccg gcgctccccc cgcatccccg agccggcagc
     1261 gtgcggggac agcccgggca cggggaaggt ggcacgggat cgctttcctc tgaacgcttc
     1321 tcgctgctct ttgagcctgc agacacctgg ggggatacgg ggaaaaggcc tccacggcca
     1381 aggatctgcg atcgctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
     1441 gagaagttgg ggggaggggt cggcaattga acgggtgcct agagaaggtg gcgcggggta
     1501 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     1561 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     1621 cagctgaagc ttcgaggggc tcgcatctct ccttcacgcg cccgccgccc tacctgaggc
     1681 cgccatccac gccggttgag tcgcgttctg ccgcctcccg cctgtggtgc ctcctgaact
     1741 gcgtccgccg tctaggtaag tttaaagctc aggtcgagac cgggcctttg tccggcgctc
     1801 ccttggagcc tacctagact cagccggctc tccacgcttt gcctgaccct gcttgctcaa
     1861 ctctacgtct ttgtttcgtt ttctgttctg cgccgttaca gatccaagct gtgaccggcg
     1921 cctactctag gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1981 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2041 gggcgatgcc acctacggca agctgaccct gaagctgatc tgcaccaccg gcaagctgcc
     2101 cgtgccctgg cccaccctcg tgaccaccct gggctacggc ctgcagtgct tcgcccgcta
     2161 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2221 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2281 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2341 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcaccgc
     2401 cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg
     2461 cggcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2521 gctgcccgac aaccactacc tgagctacca gtccaagctg agcaaagacc ccaacgagaa
     2581 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     2641 cgagctgtac aagggatatc catcacactg gcggccgctc gaggccacca tgtcaaacgt
     2701 gagagtgtct aacgggagcc cgagcctgga gcggatggac gccagacaag cggagcaccc
     2761 caagccttcc gcctgcagaa atctcttcgg cccggtcaat catgaagaac taacccggga
     2821 cttggagaag cactgccggg atatggaaga agcgagtcag cgcaagtgga atgccgacgc
     2881 ccagaatcat aagcccctgg agggcagata cgagtggcag gaggtggaga ggggcagctt
     2941 gcccgagttc tactacaggc ccccgcgccc ccccaagagc gcctgcaagg tgctggcgca
     3001 ggagagccag gatgtcagcg ggagccgcca ggcggtgcct ttaattgggt ctcaggcaaa
     3061 ctctgaggac cggcatttgg tggaccaaat gcctgactcg tcagacaatc cggctgggtt
     3121 agcggagcag tgtccaggga tgaggaagcg acctgctgca gaagattctt cttcgcaaaa
     3181 caaaagggcc aacagaacag aagaaaatgt ttcagacggt tccccgaacg ctggcactgt
     3241 ggagcagacg cccaagaagc ccggccttcg acgccagacg taagcggaat tcgaatttaa
     3301 atcggatccg cggccgcgcc cctctccctc ccccccccct aacgttactg gccgaagccg
     3361 cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt
     3421 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct
     3481 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct
     3541 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc
     3601 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc
     3661 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc
     3721 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc
     3781 tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct
     3841 aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca
     3901 caaccatggc gtccggaatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3961 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4021 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4081 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc gcgacgggcg
     4141 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4201 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4261 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4321 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4381 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4441 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4501 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4561 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4621 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4681 ccttctatcg ccttcttgac gagttcttct gagtcgacaa tcaacctctg gattacaaaa
     4741 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     4801 ctgctttaat gcctttgtat catgcgttaa ctaaacttgt ttattgcagc ttataatggt
     4861 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4921 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggaattg actcaaatga
     4981 tgtcaattag tctatcagaa gctcatctgg tctcccttcc gggggacaag acatccctgt
     5041 ttaatattta aacagcagtg ttcccaaact gggttcttat atcccttgct ctggtcaacc
     5101 aggttgcagg gtttcctgtc ctcacaggaa cgaagtccct aaagaaacag tggcagccag
     5161 gtttagcccc ggaattgact ggattccttt tttagggccc attggtatgg ctttttcccc
     5221 gtatcccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga ggaaagcgat
     5281 cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg gggatgcggg
     5341 gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta gcgggggagg
     5401 gacgtaatta catccctggg ggctttgggg gggggctgtc cctgatatct ataacaagaa
     5461 aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta
     5521 tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc
     5581 gttatagttc aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca
     5641 agcggcgact gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga
     5701 gcaatatttc aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaatcta
     5761 gctgcatcag gatcatatcg tcgggtcttt tttccggctc agtcatcgcc caagctggcg
     5821 ctatctgggc atcggggagg aagaagcccg tgccttttcc cgcgaggttg aagcggcatg
     5881 gaaagagttt gccgaggatg actgctgctg cattgacgtt gagcgaaaac gcacgtttac
     5941 catgatgatt cgggaaggtg tggccatgca cgcctttaac ggtgaactgt tcgttcaggc
     6001 cacctgggat accagttcgt cgcggctttt ccggacacag ttccggatgg tcagcccgaa
     6061 gcgcatcagc aacccgaaca ataccggcga cagccggaac tgccgtgccg gtgtgcagat
     6121 taatgacagc ggtgcggcgc tgggatatta cgtcagcgag gacgggtatc ctggctggat
     6181 gccgcagaaa tggacatgga taccccgtga gttacccggc gggcgcgctt ggcgtaatca
     6241 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     6301 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     6361 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6421 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6481 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6541 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6601 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     6661 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     6721 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     6781 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     6841 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6901 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6961 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     7021 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     7081 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     7141 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7201 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7261 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     7321 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     7381 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     7441 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     7501 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     7561 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     7621 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     7681 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     7741 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     7801 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     7861 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     7921 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7981 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     8041 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     8101 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     8161 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     8221 gcaaaaaagg gaataagggc g
//
