LOCUS       sequence-233453-        7300 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 7300)
  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 7300)
  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..7300
                     /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             1765..1818
                     /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(1819..1852)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1861..1881
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3844..3963
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3966..3999
                     /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(5112..5132)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5112..5130)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5151..5167)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5151..5167)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5164..5186)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5175..5191
                     /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(5199..5205,5206..5223,5224..5229))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5244..5265
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5382..5399)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5553..6141)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5633..5652)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6312..7103,7104..7172))
                     /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     6935..6954
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7173..7277)
                     /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 tgtgtgtgtg tctgagtgtg tgtgtacatg tgttcaggtg gctacagaag aactggtata
     1081 tgtgaccccc ccgccccgcc ccgccccgcc ccatgtattt gtcattgaaa atgtcacctt
     1141 ttcttcctta gcaaacaaat tgcttcactc ttatgctttg aagccaaata cattgtgtct
     1201 tccggctaag tggctttcac tcacaagcca gtccatacca tgcatttagc aatagttctg
     1261 gatatgtctt ctggctgatt ggcaatcctt catgggttaa ttcatgctag gtgtctaagg
     1321 gtcctagcat gtaaggacca ggagcacagc ccactgtgat acatagtatc agttccctgt
     1381 cagctgtcag caagtgtttg gagaaagcag tgggactggt agtctggaag gttcctttga
     1441 gcccaatagt cctgtcagaa ggcatgggga agggtagaga catggggcag atacagtggg
     1501 cgtgtggggg gtgggggctg ggggctggtg gctttcccag acctggaact ccagaacaca
     1561 gctgttgttg gtgtcttaga gacatggacc tttctaacct cacatctttt ctctctatac
     1621 tccaggatct tcggcttccc tgctcactac acggacgtgt ccaacatggg ccgcggcgcc
     1681 cgtcagaagc tgctgggcag gtcctggagt gtaccggtca tcagacacct gtttgccccc
     1741 ttgaaggact actttgcctg tgaagaggga agaggtagcc tgctaacttg cggggacgta
     1801 gaggaaaatc ctggtcccat aacttcgtat agcatacatt atacgaagtt atcaatgggc
     1861 ccaaaaaaga aaagaaaagt tgaattcatg gccagaaaag tggacctgac cagttgtgac
     1921 cgagagccga tacacattcc tggctctatc cagccatgtg ggtgtttgct ggcctgcgat
     1981 gcccaggcag tcaggattac tcgaattacc gaaaatgcgg gtgccttctt tggccgtgaa
     2041 acgccccgtg tcggcgaact cttagccgat tatttcggag aaaccgaggc tcacgcactt
     2101 aggaacgcac tggcgcaatc aagcgatccc aaacgccctg ctctcatctt tgggtggaga
     2161 gatggactga caggccgtac tttcgacata agccttcacc ggcatgacgg aaccagtata
     2221 attgagtttg aaccggccgc cgctgagcaa gcagacaacc cccttcgcct gaccagacaa
     2281 attatcgccc gcactaagga gctgaaatcg ctggaagaaa tggccgctcg tgttcccagg
     2341 tatcttcagg caatgctggg ttatcatagg gtaatgctct acagattcgc cgacgatggc
     2401 tctggtatgg taatagggga ggccaagcga tccgacctgg agtcttttct ggggcagcat
     2461 ttccccgcat ctctagtacc ccagcaggca aggcttttgt acctcaagaa tgccatccgc
     2521 gtggtatcgg actcccgcgg tattagtagc aggatcgttc cagaacacga cgcctctgga
     2581 gctgcgctgg atctgtcttt cgctcacctc cgatctatca gcccttgtca tttagagttt
     2641 ctgcgaaata tgggggtcag cgcaagtatg tccctgagca taataatcga tggaacacta
     2701 tggggcctaa taatttgcca tcattacgaa cctagagcag taccaatggc acagcgagtg
     2761 gccgccgaaa tgttcgccga ttttttgtct cttcatttta ctgccgctca ccatcagcgc
     2821 ggccacggga ctggctcgac aggttctggc agtagtggga ctgcttcatc agaagataat
     2881 aacatggcca tggccagaaa agtggacctg accagttgtg accgagagcc gatacacatt
     2941 cctggctcta tccagccatg tgggtgtttg ctggcctgcg atgcccaggc agtcaggatt
     3001 actcgaatta ccgaaaatgc gggtgccttc tttggccgtg aaacgccccg tgtcggcgaa
     3061 ctcttagccg attatttcgg agaaaccgag gctcacgcac ttaggaacgc actggcgcaa
     3121 tcaagcgatc ccaaacgccc tgctctcatc tttgggtgga gagatggact gacaggccgt
     3181 actttcgaca taagccttca ccggcatgac ggaaccagta taattgagtt tgaaccggcc
     3241 gccgctgagc aagcagacaa cccccttcgc ctgaccagac aaattatcgc ccgcactaag
     3301 gagctgaaat cgctggaaga aatggccgct cgtgttccca ggtatcttca ggcaatgctg
     3361 ggttatcata gggtaatgct ctacagattc gccgacgatg gctctggtat ggtaataggg
     3421 gaggccaagc gatccgacct ggagtctttt ctggggcagc atttccccgc atctctagta
     3481 ccccagcagg caaggctttt gtacctcaag aatgccatcc gcgtggtatc ggactcccgc
     3541 ggtattagta gcaggatcgt tccagaacac gacgcctctg gagctgcgct ggatctgtct
     3601 ttcgctcacc tccgatctat cagcccttgt catttagagt ttctgcgaaa tatgggggtc
     3661 agcgcaagta tgtccctgag cataataatc gatggaacac tatggggcct aataatttgc
     3721 catcattacg aacctagagc agtaccaatg gcacagcgag tggccgccga aatgttcgcc
     3781 gattttttgt ctcttcattt tactgccgct caccatcagc gcggatccgc gtttaaactc
     3841 gagtctcacg gcttccctcc cgaggtggag gagcaggccg ccggcaccct gcccatgagc
     3901 tgcgcccagg agagcggcat ggatagacac cctgctgctt gcgccagcgc caggatcaac
     3961 gtcttgaagt tcctattctc tagaaagtat aggaacttct agtagttcta cccaggactg
     4021 gggagctctc ggtcagagcc agtgcccaga gtcacccctc cctgaaggca cctcacctgt
     4081 ccccttttta gctcacctgt gtggggcctc acatcactgt acctcagctt tctcctgctc
     4141 agtgggagca gagcctcctg gcccttgcag gggagccccg gtgctccctc cgtgtgcaca
     4201 gctcagacct ggctgcttag agtagcccgg catggtgctc atgttctctt accctgaaac
     4261 tttaaaactt gaagtaggta gtaagatggc tttcttttac cctcctgagt ttatcactca
     4321 gaagtgatgg ctaagatacc aaaaaaacaa acaaaaacag aaacaaaaaa caaaaaaaaa
     4381 acctcaacag ctctcttagt actcaggttc atgctgcaaa atcacttgag attttgtttt
     4441 taagtaaccc gtgctccaca tttgctggag gatgctattg tgaatgtggg ctcagatgag
     4501 caaggtcaag gggccaaaaa aaattccccc tctcccccca ggagtatttg aagatgatgt
     4561 ttatggttta agtcttcctg gcaccttccc cttgctttgg tacaagggct gaagtcctgt
     4621 tggtcttgta gcatttccca ggatgatgat gtcagcaggg atgacatcat cacctttagg
     4681 gcttttccct ggcaggggcc catgtggcta gtcctcacga agactggagt agaatgtttg
     4741 gagctcagga agggtgggtg gagtggccct cttccaggtg tgagggatac gaaggaggaa
     4801 gcttagggaa atccattccc cactccctct tgccaaatga ggggcccagt ccccaacagc
     4861 tcaggtcccc agaaccccct agttcctcat gagaagctag gaccagaagc acatcgttcc
     4921 ccttatctga gcagtgtttg gggaactaca gtgaaaacct tctggagatg ttaaaagctt
     4981 tttaccccac gatagattgt gtttttaagg ggtgcttttt ttaggggcat cactggagat
     5041 aagaaagctg catttcagaa atgccatcgt aatgggcggc cgccaccgcg gtggagctcc
     5101 agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg
     5161 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5221 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5281 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5341 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5401 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5461 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5521 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5581 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5641 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5701 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5761 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5821 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5881 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5941 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     6001 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6061 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6121 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6181 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6241 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6301 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6361 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6421 ccatctggcc ccagtgctgc aatgataccg cggctgccac gctcaccggc tccagattta
     6481 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6541 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6601 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6661 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6721 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6781 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6841 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6901 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6961 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7021 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7081 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7141 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7201 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7261 caaatagggg ttccgcgcac atttccccga aaagtgccac
//
