LOCUS       sequence-233450-        7136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Trim28 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 7136)
  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 7136)
  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-Trim28-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7136
                     /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"
     RBS             1333..1341
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             1676..1729
                     /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(1730..1763)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1772..1792
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3755..3874
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3877..3910
                     /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(4948..4968)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4948..4966)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4987..5003)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4987..5003)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5000..5022)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5011..5027
                     /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(5035..5041,5042..5059,5060..5065))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5080..5101
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5218..5235)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5389..5977)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5469..5488)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6148..6939,6940..7008))
                     /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     6771..6790
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7009..7113)
                     /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 gagaagtggg ccaggtatcc tggatgacag tgccactatc tgccgtgtct
      721 gccagaaacc aggtgacctg gtcatgtgta accagtgcga attttgcttc cacctggatt
      781 gccacctccc tgccctgcag gatgttccag ggtatgtgtg aggctgggca ggatcagata
      841 gattgtggag agagagctgg ttgtcatctt ttacatttcc taatcttcct catcctaggg
      901 aggaatggag ttgctcactc tgccacgtgc tccctgacct aaaggaggaa gatggaagcc
      961 tcagcctgga tggagcagat agcactggtg tggtagctaa actctcacca gccaaccagc
     1021 gggtaagagc tgaatgcatg tgttgtgtgg cccagtagct gaagctttcc aagctacaac
     1081 tctgatatct cttgtattgt ctagaaatgt gagcgtgttc tcctggccct gttctgccat
     1141 gaaccatgcc gtcccttgca tcagctggct accgactcta cattctccat ggtaagtccc
     1201 agaacaggag aggttgcagg tgccaagagg tggtgacccc cctcaatgtc tatgttgtct
     1261 ctccctacag gagcagcctg gtggtaccct agacctgacc ttgattcgtg ctcgcctcca
     1321 agagaagctg tcacctcctt atagctcccc ccaggagttt gctcaagatg tgggccgcat
     1381 gttcaaacag ttcaacaagc tgactgaggt gagcctgcag aatgaagccg ggggtggggg
     1441 agtatagcag gcaggggaaa gaaaagccag gacccactca cgtgtgtatt cttaattgga
     1501 acaggacaag gcagatgttc agtccatcat cggcttgcag cgcttctttg agacacgcat
     1561 gaatgatgcc tttggtgaca ccaagttttc tgctgtgctg gtagaaccac caccattgaa
     1621 ccttcccagt gctggcctaa gttctcagga gctctctggc cctggtgatg gccccgaggg
     1681 aagaggtagc ctgctaactt gcggggacgt agaggaaaat cctggtccca taacttcgta
     1741 tagcatacat tatacgaagt tatcaatggg cccaaaaaag aaaagaaaag ttgaattcat
     1801 ggccagaaaa gtggacctga ccagttgtga ccgagagccg atacacattc ctggctctat
     1861 ccagccatgt gggtgtttgc tggcctgcga tgcccaggca gtcaggatta ctcgaattac
     1921 cgaaaatgcg ggtgccttct ttggccgtga aacgccccgt gtcggcgaac tcttagccga
     1981 ttatttcgga gaaaccgagg ctcacgcact taggaacgca ctggcgcaat caagcgatcc
     2041 caaacgccct gctctcatct ttgggtggag agatggactg acaggccgta ctttcgacat
     2101 aagccttcac cggcatgacg gaaccagtat aattgagttt gaaccggccg ccgctgagca
     2161 agcagacaac ccccttcgcc tgaccagaca aattatcgcc cgcactaagg agctgaaatc
     2221 gctggaagaa atggccgctc gtgttcccag gtatcttcag gcaatgctgg gttatcatag
     2281 ggtaatgctc tacagattcg ccgacgatgg ctctggtatg gtaatagggg aggccaagcg
     2341 atccgacctg gagtcttttc tggggcagca tttccccgca tctctagtac cccagcaggc
     2401 aaggcttttg tacctcaaga atgccatccg cgtggtatcg gactcccgcg gtattagtag
     2461 caggatcgtt ccagaacacg acgcctctgg agctgcgctg gatctgtctt tcgctcacct
     2521 ccgatctatc agcccttgtc atttagagtt tctgcgaaat atgggggtca gcgcaagtat
     2581 gtccctgagc ataataatcg atggaacact atggggccta ataatttgcc atcattacga
     2641 acctagagca gtaccaatgg cacagcgagt ggccgccgaa atgttcgccg attttttgtc
     2701 tcttcatttt actgccgctc accatcagcg cggccacggg actggctcga caggttctgg
     2761 cagtagtggg actgcttcat cagaagataa taacatggcc atggccagaa aagtggacct
     2821 gaccagttgt gaccgagagc cgatacacat tcctggctct atccagccat gtgggtgttt
     2881 gctggcctgc gatgcccagg cagtcaggat tactcgaatt accgaaaatg cgggtgcctt
     2941 ctttggccgt gaaacgcccc gtgtcggcga actcttagcc gattatttcg gagaaaccga
     3001 ggctcacgca cttaggaacg cactggcgca atcaagcgat cccaaacgcc ctgctctcat
     3061 ctttgggtgg agagatggac tgacaggccg tactttcgac ataagccttc accggcatga
     3121 cggaaccagt ataattgagt ttgaaccggc cgccgctgag caagcagaca acccccttcg
     3181 cctgaccaga caaattatcg cccgcactaa ggagctgaaa tcgctggaag aaatggccgc
     3241 tcgtgttccc aggtatcttc aggcaatgct gggttatcat agggtaatgc tctacagatt
     3301 cgccgacgat ggctctggta tggtaatagg ggaggccaag cgatccgacc tggagtcttt
     3361 tctggggcag catttccccg catctctagt accccagcag gcaaggcttt tgtacctcaa
     3421 gaatgccatc cgcgtggtat cggactcccg cggtattagt agcaggatcg ttccagaaca
     3481 cgacgcctct ggagctgcgc tggatctgtc tttcgctcac ctccgatcta tcagcccttg
     3541 tcatttagag tttctgcgaa atatgggggt cagcgcaagt atgtccctga gcataataat
     3601 cgatggaaca ctatggggcc taataatttg ccatcattac gaacctagag cagtaccaat
     3661 ggcacagcga gtggccgccg aaatgttcgc cgattttttg tctcttcatt ttactgccgc
     3721 tcaccatcag cgcggatccg cgtttaaact cgagtctcac ggcttccctc ccgaggtgga
     3781 ggagcaggcc gccggcaccc tgcccatgag ctgcgcccag gagagcggca tggatagaca
     3841 ccctgctgct tgcgccagcg ccaggatcaa cgtcttgaag ttcctattct ctagaaagta
     3901 taggaacttc tagtgaagct ggggctcttg tggtcagccc agtccagctc tggtctctgt
     3961 attttcaccc cataccctgt cctttggtgg cctgactcct gttcttgctg gccccatcgt
     4021 cccctcagtc cctcttcaca aaatggtttt tacttctgtg gatttaataa aaacttcact
     4081 gagtcagttg ctcatgcctc agctgtctgg ggactgttcc tgaggtttta agactcattc
     4141 tacaggtgac cttacttccc tctttttggc caggtgaaac atttgtacac tgtccttggt
     4201 aagacttgga tggagcccgg cgtggtggtg cacgccttta atcccagcac ttgggaggca
     4261 gaggcagccg gatttctgag ttccaggcca gcctggtcta caaagtgagt tccaggagcc
     4321 agggctatac agagaaactc tgagacagag tttggatgga ggcctcaggt gtgcagtgcc
     4381 tcccagtggc cagatacaaa tttagacacg aagcaactta actgcttaca tttaatcaac
     4441 tgcttacatt taatcaaagt gggttgtgtg agattctcaa agggtttttt tgttttttta
     4501 aatattattt cttcagacac tccaagaaga gggcatcaga tctcattaca gatggttgtg
     4561 agccaccatg tggttgctgg gatttgaact tatgaccttt ggaagagcag tcagtgcttt
     4621 taaagcgctg agcccccctc aaagggttgt ttttacagac attgtctggc tatcctggac
     4681 atctgtaaat gaggctggct tttaactcag atctacctct agagtgccag gattaaaggt
     4741 gtgcattagc actcaaattt agcatagcat tcctttctat gacccataac tgagtttcat
     4801 atcccaagtt ggtggacagt taccacttta aatctcatct gttgccatgg attgtttggt
     4861 tagtatgtgt taggtggaga ggtatatatg cattaactgg atgctgcgcc cgcggccgcc
     4921 accgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     4981 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5041 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5101 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5161 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5221 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5281 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5341 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5401 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5461 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5521 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5581 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5641 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5701 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5761 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5821 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5881 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5941 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6001 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6061 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6121 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6181 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6241 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcggc tgccacgctc
     6301 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6361 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6421 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6481 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6541 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6601 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6661 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6721 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6781 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6841 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6901 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6961 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7021 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7081 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac
//
