LOCUS       sequence-233464-        7103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Peg3 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 7103)
  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 7103)
  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-Peg3-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7103
                     /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             1686..1739
                     /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(1740..1773)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1782..1802
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3765..3884
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3887..3920
                     /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(4915..4935)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4915..4933)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4954..4970)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4954..4970)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4967..4989)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4978..4994
                     /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(5002..5008,5009..5026,5027..5032))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5047..5068
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5185..5202)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5356..5944)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5436..5455)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6115..6906,6907..6975))
                     /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     6738..6757
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6976..7080)
                     /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 tgggtaccac
      661 agcacagaga taatgaaata atggaacaga gtgagctttc agatgagatt ttcattcaag
      721 gcctagccct cactgagtat caggggagtg aaacagaaga gaagcttttc gagtgcacaa
      781 tctgtgggga atgcttcttc actgccaaac agctcgggga ccaccacacc aaagttcaca
      841 aggatgagcc ctatgagtat gggccctcct acacccatgc ctcctttctc accgagcccc
      901 tcaggaagca catcccactg tacgaatgca aagattgcgg ccagtccttc ctagacgaca
      961 ctgtcatcgc tgagcgcatg gtgtttcatc ctgagcgaga aggtgggtca gaaatagtag
     1021 ctgccactgc ccaagaggtc gaagccaatg tcctcatccc acaagaagta ctgcgaatcc
     1081 aggggtcaaa tgcagaagct gctgagcccg aagtggaggc tgcagagccc gaggtggagg
     1141 ctgcagagcc tgaggtggag gctgcagagc ctaatggaga ggctgaaggg ccagatggag
     1201 aagctgctga gcctgatggc gaggctgagc agcccaatgg agaggctgaa cagccaaacg
     1261 gtgatgctga cgagccagac ggagccggga tcgaagaccc agaagagaga gctgacgagc
     1321 ctgaggaaga cgtcgaagag ccagagggag atgcagatga gcccgatggt gcagacattg
     1381 aagacccaga agaggaagga gaagatcaag agattgaggt tgaagaacca tactacaact
     1441 gtcatgaatg cgcagaaacg ttcgcttcca gctcagcctt tggcgagcat ctgaaaagtc
     1501 acgccagtgt gatcatcttc gagccggcca atgctcctgg agagtgctct ggctacattg
     1561 aacgggccag caccagtgca ggtggtgcgg agcaggcaga cgacaagtac ttcaaatgtg
     1621 atgtgtgcgg gcaactcttc aacgaccgcc tctcccttgc cagacaccag aattctcaca
     1681 ctggtgaggg aagaggtagc ctgctaactt gcggggacgt agaggaaaat cctggtccca
     1741 taacttcgta tagcatacat tatacgaagt tatcaatggg cccaaaaaag aaaagaaaag
     1801 ttgaattcat ggccagaaaa gtggacctga ccagttgtga ccgagagccg atacacattc
     1861 ctggctctat ccagccatgt gggtgtttgc tggcctgcga tgcccaggca gtcaggatta
     1921 ctcgaattac cgaaaatgcg ggtgccttct ttggccgtga aacgccccgt gtcggcgaac
     1981 tcttagccga ttatttcgga gaaaccgagg ctcacgcact taggaacgca ctggcgcaat
     2041 caagcgatcc caaacgccct gctctcatct ttgggtggag agatggactg acaggccgta
     2101 ctttcgacat aagccttcac cggcatgacg gaaccagtat aattgagttt gaaccggccg
     2161 ccgctgagca agcagacaac ccccttcgcc tgaccagaca aattatcgcc cgcactaagg
     2221 agctgaaatc gctggaagaa atggccgctc gtgttcccag gtatcttcag gcaatgctgg
     2281 gttatcatag ggtaatgctc tacagattcg ccgacgatgg ctctggtatg gtaatagggg
     2341 aggccaagcg atccgacctg gagtcttttc tggggcagca tttccccgca tctctagtac
     2401 cccagcaggc aaggcttttg tacctcaaga atgccatccg cgtggtatcg gactcccgcg
     2461 gtattagtag caggatcgtt ccagaacacg acgcctctgg agctgcgctg gatctgtctt
     2521 tcgctcacct ccgatctatc agcccttgtc atttagagtt tctgcgaaat atgggggtca
     2581 gcgcaagtat gtccctgagc ataataatcg atggaacact atggggccta ataatttgcc
     2641 atcattacga acctagagca gtaccaatgg cacagcgagt ggccgccgaa atgttcgccg
     2701 attttttgtc tcttcatttt actgccgctc accatcagcg cggccacggg actggctcga
     2761 caggttctgg cagtagtggg actgcttcat cagaagataa taacatggcc atggccagaa
     2821 aagtggacct gaccagttgt gaccgagagc cgatacacat tcctggctct atccagccat
     2881 gtgggtgttt gctggcctgc gatgcccagg cagtcaggat tactcgaatt accgaaaatg
     2941 cgggtgcctt ctttggccgt gaaacgcccc gtgtcggcga actcttagcc gattatttcg
     3001 gagaaaccga ggctcacgca cttaggaacg cactggcgca atcaagcgat cccaaacgcc
     3061 ctgctctcat ctttgggtgg agagatggac tgacaggccg tactttcgac ataagccttc
     3121 accggcatga cggaaccagt ataattgagt ttgaaccggc cgccgctgag caagcagaca
     3181 acccccttcg cctgaccaga caaattatcg cccgcactaa ggagctgaaa tcgctggaag
     3241 aaatggccgc tcgtgttccc aggtatcttc aggcaatgct gggttatcat agggtaatgc
     3301 tctacagatt cgccgacgat ggctctggta tggtaatagg ggaggccaag cgatccgacc
     3361 tggagtcttt tctggggcag catttccccg catctctagt accccagcag gcaaggcttt
     3421 tgtacctcaa gaatgccatc cgcgtggtat cggactcccg cggtattagt agcaggatcg
     3481 ttccagaaca cgacgcctct ggagctgcgc tggatctgtc tttcgctcac ctccgatcta
     3541 tcagcccttg tcatttagag tttctgcgaa atatgggggt cagcgcaagt atgtccctga
     3601 gcataataat cgatggaaca ctatggggcc taataatttg ccatcattac gaacctagag
     3661 cagtaccaat ggcacagcga gtggccgccg aaatgttcgc cgattttttg tctcttcatt
     3721 ttactgccgc tcaccatcag cgcggatccg cgtttaaact cgagtctcac ggcttccctc
     3781 ccgaggtgga ggagcaggcc gccggcaccc tgcccatgag ctgcgcccag gagagcggca
     3841 tggatagaca ccctgctgct tgcgccagcg ccaggatcaa cgtcttgaag ttcctattct
     3901 ctagaaagta taggaacttc tagtgagtaa ccaggctgaa gaaaagaaga gcaaagccaa
     3961 accttcttcc cagaaccaga cccttaataa atcacaaaga gagcctaaac caacccataa
     4021 tgtctataag aaattcacct tcctgtatac ataccggact tcacatcaaa gactttcact
     4081 ctcatcacag actgaaaaaa gaaaagacat tgaacgcagg gactctttca gttttagctg
     4141 ttccctatgg aacatcagtg tatatttggg aaagctagag tgaacatcta catcttccat
     4201 ttcatctaag taactagatt gagggaaaac ctagtgacaa ttccagacca cagaggttgc
     4261 cccagtcgac tgtaaatgat acccctttca taccctatac ataatgattc ctgccatgta
     4321 tataaatgag caaatcagtg atacatatat ttggatttag tgtgctatag aatttacagt
     4381 ttactctaca gagctaccta gcctggtact ctgatttttt ccctgaggag gaagagagca
     4441 acaatttagc atatatttgt aagtattgtc catgcagaag cttttctgtg catcatttga
     4501 accccattag tatcctttcc agtaatggag tgttctgtcc cctacctctt agatagtcct
     4561 gtgaaggtgt gggtgtgaaa gatcgtgtgt ctttgaatcc tggctgtgtg gaaacaggca
     4621 ttttagcttc tacagccatt tggtgtgcac ccagacccct tgagactgat tgtgtaaccc
     4681 tttacaatat atggatttgt ctctgtgacc caaatcaacc catccctaca tttatatacc
     4741 ttacagtggt tttcttgcaa aaaaaaaaaa tgttacattt gaggtgttct ttaaggtttg
     4801 ggtttttgtt ttctaaccaa ctcatcgtgt gtttgatagt gaattcatga aacctgaagc
     4861 ttttccctgt aaatcttaat gtctttgcct ttaaaggagc tccagctttt gttcccttta
     4921 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4981 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5041 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5101 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5161 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5221 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5281 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5341 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5401 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5461 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5521 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5581 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5641 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5701 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5761 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     5821 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5881 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5941 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6001 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6061 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6121 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6181 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6241 tgcaatgata ccgcggctgc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6301 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6361 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6421 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6481 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6541 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6601 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6661 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6721 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6781 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6841 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6901 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6961 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7021 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7081 cacatttccc cgaaaagtgc cac
//
