LOCUS       sequence-238904-        7298 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Dnmt3b 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 7298)
  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 7298)
  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-Dnmt3b-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7298
                     /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             1763..1816
                     /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(1817..1850)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1859..1879
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3842..3961
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3964..3997
                     /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(5111..5131)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5111..5129)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5150..5166)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5150..5166)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5163..5185)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5174..5190
                     /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(5198..5204,5205..5222,5223..5228))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5243..5264
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5381..5398)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5552..6140)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5632..5651)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6311..7102,7103..7171))
                     /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     6934..6953
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7172..7262)
                     /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 gagtgacaat cttaggttcc tcttctacaa aggtccctga gccttggggc
      721 tgtataagtt gtaattcgta ggattctgtc atatgtccat gttgtaggct gtcctgggat
      781 ttgagtgttt ctcagatatt gattggtact gtgtagatcc cgggaatagc aaggcctgcg
      841 cgcttgaggt gtctgcacgg tacaggtgac tccagagctg gtgtcatcct gtgatacctg
      901 ggaacagcgg gtcaggtttt ctttgtctct gggattagtt ttctctgata ttttaaacag
      961 aaacacagag agagacttgt gtatatgtgt tttgtgtatc tgaatgtgtt tgacaatgtg
     1021 tgtgtgtgtc tgagtgtgtg tgtacatgtg ttcaggtggc tacagaagaa ctggtatatg
     1081 tgaccccccc gccccgcccc gccccgcccc atgtatttgt cattgaaaat gtcacctttt
     1141 cttccttagc aaacaaattg cttcactctt atgctttgaa gccaaataca ttgtgtcttc
     1201 cggctaagtg gctttcactc acaagccagt ccataccatg catttagcaa tagttctgga
     1261 tatgtcttct ggctgattgg caatccttca tgggttaatt catgctaggt gtctaagggt
     1321 cctagcatgt aaggaccagg agcacagccc actgtgatac atagtatcag ttccctgtca
     1381 gctgtcagca aatgtttgga gaaagcagtg ggactggtag tctggaaggt tcctttgagc
     1441 ccaatagtcc tgtcagaagg catggggaag ggtagagaca tggggcagat acagtgggcg
     1501 tgtggggggt gggggctggg ggctggtggc tttcccagac ctggaactcc agaacacagc
     1561 tgttgttggt gtcttagaga catggacctt tctaacctca catcttttct ctctatactc
     1621 caggatcttc ggcttccctg ctcactacac ggacgtgtcc aacatgggcc gcggcgcccg
     1681 tcagaagctg ctgggcaggt cctggagtgt accggtcatc agacacctgt ttgccccctt
     1741 gaaggactac tttgcctgtg aagagggaag aggtagcctg ctaacttgcg gggacgtaga
     1801 ggaaaatcct ggtcccataa cttcgtatag catacattat acgaagttat caatgggccc
     1861 aaaaaagaaa agaaaagttg aattcatggc cagaaaagtg gacctgacca gttgtgaccg
     1921 agagccgata cacattcctg gctctatcca gccatgtggg tgtttgctgg cctgcgatgc
     1981 ccaggcagtc aggattactc gaattaccga aaatgcgggt gccttctttg gccgtgaaac
     2041 gccccgtgtc ggcgaactct tagccgatta tttcggagaa accgaggctc acgcacttag
     2101 gaacgcactg gcgcaatcaa gcgatcccaa acgccctgct ctcatctttg ggtggagaga
     2161 tggactgaca ggccgtactt tcgacataag ccttcaccgg catgacggaa ccagtataat
     2221 tgagtttgaa ccggccgccg ctgagcaagc agacaacccc cttcgcctga ccagacaaat
     2281 tatcgcccgc actaaggagc tgaaatcgct ggaagaaatg gccgctcgtg ttcccaggta
     2341 tcttcaggca atgctgggtt atcatagggt aatgctctac agattcgccg acgatggctc
     2401 tggtatggta ataggggagg ccaagcgatc cgacctggag tcttttctgg ggcagcattt
     2461 ccccgcatct ctagtacccc agcaggcaag gcttttgtac ctcaagaatg ccatccgcgt
     2521 ggtatcggac tcccgcggta ttagtagcag gatcgttcca gaacacgacg cctctggagc
     2581 tgcgctggat ctgtctttcg ctcacctccg atctatcagc ccttgtcatt tagagtttct
     2641 gcgaaatatg ggggtcagcg caagtatgtc cctgagcata ataatcgatg gaacactatg
     2701 gggcctaata atttgccatc attacgaacc tagagcagta ccaatggcac agcgagtggc
     2761 cgccgaaatg ttcgccgatt ttttgtctct tcattttact gccgctcacc atcagcgcgg
     2821 ccacgggact ggctcgacag gttctggcag tagtgggact gcttcatcag aaaataataa
     2881 catggccatg gccagaaaag tggacctgac cagttgtgac cgagagccga tacacattcc
     2941 tggctctatc cagccatgtg ggtgtttgct ggcctgcgat gcccaggcag tcaggattac
     3001 tcgaattacc gaaaatgcgg gtgccttctt tggccgtgaa acgccccgtg tcggcgaact
     3061 cttagccgat tatttcggag aaaccgaggc tcacgcactt aggaacgcac tggcgcaatc
     3121 aagcgatccc aaacgccctg ctctcatctt tgggtggaga gatggactga caggccgtac
     3181 tttcgacata agccttcacc ggcatgacgg aaccagtata attgagtttg aaccggccgc
     3241 cgctgagcaa gcagacaacc cccttcgcct gaccagacaa attatcgccc gcactaagga
     3301 gctgaaatcg ctggaagaaa tggccgctcg tgttcccagg tatcttcagg caatgctggg
     3361 ttatcatagg gtaatgctct acagattcgc cgacgatggc tctggtatgg taatagggga
     3421 ggccaagcga tccgacctgg agtcttttct ggggcagcat ttccccgcat ctctagtacc
     3481 ccagcaggca aggcttttgt acctcaagaa tgccatccgc gtggtatcgg actcccgcgg
     3541 tattagtagc aggatcgttc cagaacacga cgcctctgga gctgcgctgg atctgtcttt
     3601 cgctcacctc cgatctatca gcccttgtca tttagagttt ctgcgaaata tgggggtcag
     3661 cgcaagtatg tccctgagca taataatcga tggaacacta tggggcctaa taatttgcca
     3721 tcattacgaa cctagagcag taccaatggc acagcgagtg gccgccgaaa tgttcgccga
     3781 ttttttgtct cttcatttta ctgccgctca ccatcagcgc ggatccgcgt ttaaactcga
     3841 gtctcacggc ttccctcccg aggtggagga gcaggccgcc ggcaccctgc ccatgagctg
     3901 cgcccaggag agcggcatgg atagacaccc tgctgcttgc gccagcgcca ggatcaacgt
     3961 cttgaagttc ctattctcta gaaagtatag gaacttctag tagttctacc caggactggg
     4021 gagctctcgg tcagagccag tgcccagagt cacccctccc tgaaggcacc tcacctgtcc
     4081 cctttttagc tcacctgtgt ggggcctcac atcactgtac ctcagctttc tcctgctcag
     4141 tgggagcaga gcctcctggc ccttgcaggg gagccccggt gctccctccg tgtgcacagc
     4201 tcagacctgg ctgcttagag tagcccggca tggtgctcat gttctcttac cctgaaactt
     4261 taaaacttga agtaggtagt aagatggctt tcttttaccc tcctgagttt atcactcaga
     4321 agtgatggct aagataccaa aaaaacaaac aaaaacagaa acaaaaaaca aaaaaaaaaa
     4381 cctcaacagc tctcttagta ctcaggttca tgctgcaaaa tcacttgaga ttttgttttt
     4441 aagtaacccg tgctccacat ttgctggagg atgctattgt gaatgtgggc tcagatgagc
     4501 aaggtcaagg ggccaaaaaa aattccccct ctccccccag gagtatttga agatgatgtt
     4561 tatggtttaa gtcttcctgg caccttcccc ttgctttggt acaagggctg aagtcctgtt
     4621 ggtcttgtag catttcccag gatgatgatg tcagcaggga tgacatcatc acctttaggg
     4681 cttttccctg gcaggggccc atgtggctag tcctcacgaa gactggagta gaatgtttgg
     4741 agctcaggaa gggtgggtgg agtggccctc ttccaggtgt gagggatacg aaggaggaag
     4801 cttagggaaa tccattcccc actccctctt gccaaatgag gggcccagtc cccaacagct
     4861 caggtcccca gaacccccta gttcctcatg agaagctagg accagaagca catcgttccc
     4921 cttatctgag cagtgtttgg ggaactacag tgaaaacctt ctggagatgt taaaagcttt
     4981 ttaccccacg atagattgtg tttttaaggg gtgctttttt taggggcatc actggagata
     5041 agaaagctgc atttcagaaa tgccatcgta atgggcggcc gccaccgcgg tggagctcca
     5101 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     5161 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5221 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5281 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5341 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5401 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5461 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5521 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5581 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5641 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5701 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5761 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5821 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5881 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5941 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6001 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6061 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6121 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6181 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6241 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6301 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6361 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6421 catctggccc cagtgctgca atgataccgc ggctgccacg ctcaccggct ccagatttat
     6481 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6541 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6601 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6661 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6721 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6781 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6841 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6901 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6961 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7021 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7081 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7141 taagggcgac acggaaatgt tgaatactca tactcttcct tttcaatatt attgaagcat
     7201 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7261 aatagggatt ccgcgcacat ttccccgaaa agtgccac
//
