LOCUS       sequence-238847-        7140 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Rif1 to insert 2A-tdiRFP670-PEST into 
            immediately upstream of stop codon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7140)
  AUTHORS   Ochiai H, Hayashi T, Umeda M, Yoshimura M, Harada A, Shimizu Y, 
            Nakano K, Saitoh N, Liu Z, Yamamoto T, Okamura T, Ohkawa Y, Kimura 
            H, Nikaido I
  TITLE     Genome-wide kinetic properties of transcriptional bursting in mouse 
            embryonic stem cells.
  JOURNAL   Sci Adv. 2020 Jun 17;6(25):eaaz6699. doi: 10.1126/sciadv.aaz6699. 
            eCollection 2020 Jun.
  PUBMED    32596448
REFERENCE   2  (bases 1 to 7140)
  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: "Sci Adv."; 
            date: "2020-06-17"; volume: "6(25):eaaz6699. doi"; pages: " 
            10.1126/sciadv.aaz6699. eCollection 2020 Jun"
COMMENT     Sequence Label: pTV-mRif1-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1693..1746
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    complement(1747..1780)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1789..1809
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3772..3891
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3894..3927
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(4953..4973)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4953..4971)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4992..5008)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4992..5008)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5005..5027)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5016..5032
                     /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(5040..5046,5047..5064,5065..5070))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5085..5106
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5223..5240)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5394..5982)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5474..5493)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6153..6944,6945..7013))
                     /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     6776..6795
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7014..7104)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gagtggggca tttcatgttt catgtaattc ctgtttaaaa tatgttttat
      721 tctgtttgct actgttttaa atgcataatc aagatgaaat ctcgtggact tgaagaaatt
      781 ccaatttttg atatttctga gaaggcagta aatggagtgg agagcaggac tgtctccacg
      841 gatgaagaaa ggttcgcctc aggtatgcta gccaagcacg ttagtttcat atggaataag
      901 tgttcttggt aaacataact ttgttaatgg ttttacatct ttttaaaaaa tgttattgta
      961 gtatatacac tttgtttgta tgtatttgtg tactttggtt cctctggaac taatattaga
     1021 gcagttgtga tttgccgtgt gattgcctgg aaatgaatcc tggtcctttg gaaggataga
     1081 cttttaagta ctgagatatt tctccaggcc aatgctttaa ttttaatttg accacaacta
     1141 ttaattcacc atattcccct gcttccctcc cctccaccaa gtacatgagg ggaataccta
     1201 tttaatttta agtgtgtcta actggttttc tctgtgttag tgtttagtcc atcaaggcat
     1261 agataagtat ggtaacattt tgttcagtta ttttatgcag agtaacaagc tatggtagat
     1321 tttacttgca ttgtgttttc ttgatcaagt ttctcatagc ttatttcctc tcagtatgat
     1381 tggagataca gatcagtttt tataactggt atggatttaa tatatatata tatatatata
     1441 tatntgtgtg tgtgtgtgtg tggctttttt tttcctctta tagatttgat tgaacctgtg
     1501 accctagata ccccattgtc taaaaatctt gtggcacaaa ttagtgctct tgctcttcaa
     1561 ctggactcag aagatctcta cagttacact ggaagccagc tatttgaaat gcatgaaaaa
     1621 cttggtacca tggcaaactc tataataaga aatctacagt cacgttggag atcaccagcc
     1681 catgaaaatt ctgagggaag aggtagcctg ctaacttgcg gggacgtaga ggaaaatcct
     1741 ggtcccataa cttcgtatag catacattat acgaagttat caatgggccc aaaaaagaaa
     1801 agaaaagttg aattcatggc cagaaaagtg gacctgacca gttgtgaccg agagccgata
     1861 cacattcctg gctctatcca gccatgtggg tgtttgctgg cctgcgatgc ccaggcagtc
     1921 aggattactc gaattaccga aaatgcgggt gccttctttg gccgtgaaac gccccgtgtc
     1981 ggcgaactct tagccgatta tttcggagaa accgaggctc acgcacttag gaacgcactg
     2041 gcgcaatcaa gcgatcccaa acgccctgct ctcatctttg ggtggagaga tggactgaca
     2101 ggccgtactt tcgacataag ccttcaccgg catgacggaa ccagtataat tgagtttgaa
     2161 ccggccgccg ctgagcaagc agacaacccc cttcgcctga ccagacaaat tatcgcccgc
     2221 actaaggagc tgaaatcgct ggaagaaatg gccgctcgtg ttcccaggta tcttcaggca
     2281 atgctgggtt atcatagggt aatgctctac agattcgccg acgatggctc tggtatggta
     2341 ataggggagg ccaagcgatc cgacctggag tcttttctgg ggcagcattt ccccgcatct
     2401 ctagtacccc agcaggcaag gcttttgtac ctcaagaatg ccatccgcgt ggtatcggac
     2461 tcccgcggta ttagtagcag gatcgttcca gaacacgacg cctctggagc tgcgctggat
     2521 ctgtctttcg ctcacctccg atctatcagc ccttgtcatt tagagtttct gcgaaatatg
     2581 ggggtcagcg caagtatgtc cctgagcata ataatcgatg gaacactatg gggcctaata
     2641 atttgccatc attacgaacc tagagcagta ccaatggcac agcgagtggc cgccgaaatg
     2701 ttcgccgatt ttttgtctct tcattttact gccgctcacc atcagcgcgg ccacgggact
     2761 ggctcgacag gttctggcag tagtgggact gcttcatcag aaaataataa catggccatg
     2821 gccagaaaag tggacctgac cagttgtgac cgagagccga tacacattcc tggctctatc
     2881 cagccatgtg ggtgtttgct ggcctgcgat gcccaggcag tcaggattac tcgaattacc
     2941 gaaaatgcgg gtgccttctt tggccgtgaa acgccccgtg tcggcgaact cttagccgat
     3001 tatttcggag aaaccgaggc tcacgcactt aggaacgcac tggcgcaatc aagcgatccc
     3061 aaacgccctg ctctcatctt tgggtggaga gatggactga caggccgtac tttcgacata
     3121 agccttcacc ggcatgacgg aaccagtata attgagtttg aaccggccgc cgctgagcaa
     3181 gcagacaacc cccttcgcct gaccagacaa attatcgccc gcactaagga gctgaaatcg
     3241 ctggaagaaa tggccgctcg tgttcccagg tatcttcagg caatgctggg ttatcatagg
     3301 gtaatgctct acagattcgc cgacgatggc tctggtatgg taatagggga ggccaagcga
     3361 tccgacctgg agtcttttct ggggcagcat ttccccgcat ctctagtacc ccagcaggca
     3421 aggcttttgt acctcaagaa tgccatccgc gtggtatcgg actcccgcgg tattagtagc
     3481 aggatcgttc cagaacacga cgcctctgga gctgcgctgg atctgtcttt cgctcacctc
     3541 cgatctatca gcccttgtca tttagagttt ctgcgaaata tgggggtcag cgcaagtatg
     3601 tccctgagca taataatcga tggaacacta tggggcctaa taatttgcca tcattacgaa
     3661 cctagagcag taccaatggc acagcgagtg gccgccgaaa tgttcgccga ttttttgtct
     3721 cttcatttta ctgccgctca ccatcagcgc ggatccgcgt ttaaactcga gtctcacggc
     3781 ttccctcccg aggtggagga gcaggccgcc ggcaccctgc ccatgagctg cgcccaggag
     3841 agcggcatgg atagacaccc tgctgcttgc gccagcgcca ggatcaacgt cttgaagttc
     3901 ctattctcta gaaagtatag gaacttctag tagtctttta agagaaatgt aaagtttttt
     3961 caccatcaga ttttattttt tggcttttag aaatattcac accatttaaa gaggacactt
     4021 tgtaaaattg ataaaataat caaatcctga aaggtaatga attactaggt catttttata
     4081 agttcattag atcttcccta ctgtattttc ttggccagta cacatagttt tttaagaagt
     4141 ttaaatgtta ctgaaacttg aaagcaaaag agatatacat accttttttt tttcatgctt
     4201 taattcatgc atattttggt atagctcatg taaaagattt taattaagat agaagaaaag
     4261 ttttatttac actttattga gttgacagag atgtttatac tctttcaaaa tatttatatt
     4321 tgttgtgcct ggtggaacac ctgtactgag atgcaaaaga atgagaacat ttcctgaaga
     4381 ttacagctgt agaaaatctt tgaaatatca agtttcaact tgtgtgttaa aataataatg
     4441 cttaattcac taagattctg ttcattttgc attgacaaat acttgtgcag cagcctttgt
     4501 gaaaatgtgt tcttatgaag gtatagtgtt caaccttaaa tgcatgactc tgtatattat
     4561 gtatagaaga caatgttttt taatttataa agttcagcct tcattaatcg catgggtttt
     4621 tctgcagctt tttgtgaata ctattgtttt gtgtaataga aacatttgta tatatttaat
     4681 atcaaaatat ccatgtggat agtaaaagta cttcattgga cctactgtgg atatttgtag
     4741 gagtctttat ctacaaataa agcaacagat agttttgtat ttagttaaaa catgagttta
     4801 ttttctcaaa tgtccagaat aattaaaatt attggaacca gcaaatgtta cataccactg
     4861 ccttttttct tatggtttgg aagaggatcc cagccactag aaaatgtgcc tccctcgcgg
     4921 ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
     4981 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5041 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5101 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5161 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5221 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5281 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5341 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5401 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5461 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5521 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5581 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5641 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5701 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5761 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5821 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5881 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5941 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6001 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6061 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6121 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6181 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6241 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcggctgcca
     6301 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6361 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6421 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6481 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6541 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6601 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6661 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6721 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6781 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6841 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6901 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6961 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7021 cttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7081 aatgtattta gaaaaataaa caaataggga ttccgcgcac atttccccga aaagtgccac
//
