LOCUS       sequence-233461-        7037 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Trim27 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 7037)
  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 7037)
  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-Trim27-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7037
                     /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             1593..1646
                     /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(1647..1680)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1689..1709
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3672..3791
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3794..3827
                     /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(4849..4869)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4849..4867)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4888..4904)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4888..4904)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4901..4923)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4912..4928
                     /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(4936..4942,4943..4960,4961..4966))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4981..5002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5119..5136)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5290..5878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5370..5389)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6049..6840,6841..6909))
                     /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     6672..6691
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6910..7014)
                     /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 tgggtacctt
      661 gtattacttt cattgttatc catacagagg caatcagcta atctaaccgt aaatttttag
      721 gttctgagtc agggtcttac tatgtaacac tgaccatgca acatgggctg gtctggaact
      781 cctgaagata acttctgcct cagtctccac gtgtgccacc atgcttagtt ccaagagaat
      841 ttttttacat aatcagttca ccgtgtgtct gatatataat gagtacagcc tctggtgggc
      901 atctcctaca gtgttctggg gaagtcagtg aaataccagg tagtcttgag tgggctcttc
      961 tggccctcag atgacacccc cccccctttg tttctacagt tgatgtaacc ctggacccag
     1021 acacagccta tcccagtctg attctttctg ataatcttcg gcaagtacgg tacagttacc
     1081 tccagcagga tcttccagac aaccctgagc gattcaatct gtttccctgt gtcttgggct
     1141 ctccatgttt catcgctggg agacattact gggaggtaga ggtgggagat aaagccaagt
     1201 ggaccatagg tgtctgtgaa gactcggtgt gcagaaaagg tggggtgacc tcagcccccc
     1261 agaatggatt ctgggcagtg tcattgtggt atgggaaaga atattgggct cttacatccc
     1321 caatgactgc actacccctg cggacccctc tccagagagt agggattttc ttggactatg
     1381 atgctggtga ggtttccttc tacaatgtga cagagagatg ccataccttt actttctctc
     1441 acgctacctt ctgtgggcca gtccggccct acttcagcct aagctactcg ggagggaaga
     1501 gcgcagctcc tttgatcatc tgccctatga gtgggattga tgggttttct ggccatgttg
     1561 ggaatcatgg tcattccatg gagacctctc cggagggaag aggtagcctg ctaacttgcg
     1621 gggacgtaga ggaaaatcct ggtcccataa cttcgtatag catacattat acgaagttat
     1681 caatgggccc aaaaaagaaa agaaaagttg aattcatggc cagaaaagtg gacctgacca
     1741 gttgtgaccg agagccgata cacattcctg gctctatcca gccatgtggg tgtttgctgg
     1801 cctgcgatgc ccaggcagtc aggattactc gaattaccga aaatgcgggt gccttctttg
     1861 gccgtgaaac gccccgtgtc ggcgaactct tagccgatta tttcggagaa accgaggctc
     1921 acgcacttag gaacgcactg gcgcaatcaa gcgatcccaa acgccctgct ctcatctttg
     1981 ggtggagaga tggactgaca ggccgtactt tcgacataag ccttcaccgg catgacggaa
     2041 ccagtataat tgagtttgaa ccggccgccg ctgagcaagc agacaacccc cttcgcctga
     2101 ccagacaaat tatcgcccgc actaaggagc tgaaatcgct ggaagaaatg gccgctcgtg
     2161 ttcccaggta tcttcaggca atgctgggtt atcatagggt aatgctctac agattcgccg
     2221 acgatggctc tggtatggta ataggggagg ccaagcgatc cgacctggag tcttttctgg
     2281 ggcagcattt ccccgcatct ctagtacccc agcaggcaag gcttttgtac ctcaagaatg
     2341 ccatccgcgt ggtatcggac tcccgcggta ttagtagcag gatcgttcca gaacacgacg
     2401 cctctggagc tgcgctggat ctgtctttcg ctcacctccg atctatcagc ccttgtcatt
     2461 tagagtttct gcgaaatatg ggggtcagcg caagtatgtc cctgagcata ataatcgatg
     2521 gaacactatg gggcctaata atttgccatc attacgaacc tagagcagta ccaatggcac
     2581 agcgagtggc cgccgaaatg ttcgccgatt ttttgtctct tcattttact gccgctcacc
     2641 atcagcgcgg ccacgggact ggctcgacag gttctggcag tagtgggact gcttcatcag
     2701 aagataataa catggccatg gccagaaaag tggacctgac cagttgtgac cgagagccga
     2761 tacacattcc tggctctatc cagccatgtg ggtgtttgct ggcctgcgat gcccaggcag
     2821 tcaggattac tcgaattacc gaaaatgcgg gtgccttctt tggccgtgaa acgccccgtg
     2881 tcggcgaact cttagccgat tatttcggag aaaccgaggc tcacgcactt aggaacgcac
     2941 tggcgcaatc aagcgatccc aaacgccctg ctctcatctt tgggtggaga gatggactga
     3001 caggccgtac tttcgacata agccttcacc ggcatgacgg aaccagtata attgagtttg
     3061 aaccggccgc cgctgagcaa gcagacaacc cccttcgcct gaccagacaa attatcgccc
     3121 gcactaagga gctgaaatcg ctggaagaaa tggccgctcg tgttcccagg tatcttcagg
     3181 caatgctggg ttatcatagg gtaatgctct acagattcgc cgacgatggc tctggtatgg
     3241 taatagggga ggccaagcga tccgacctgg agtcttttct ggggcagcat ttccccgcat
     3301 ctctagtacc ccagcaggca aggcttttgt acctcaagaa tgccatccgc gtggtatcgg
     3361 actcccgcgg tattagtagc aggatcgttc cagaacacga cgcctctgga gctgcgctgg
     3421 atctgtcttt cgctcacctc cgatctatca gcccttgtca tttagagttt ctgcgaaata
     3481 tgggggtcag cgcaagtatg tccctgagca taataatcga tggaacacta tggggcctaa
     3541 taatttgcca tcattacgaa cctagagcag taccaatggc acagcgagtg gccgccgaaa
     3601 tgttcgccga ttttttgtct cttcatttta ctgccgctca ccatcagcgc ggatccgcgt
     3661 ttaaactcga gtctcacggc ttccctcccg aggtggagga gcaggccgcc ggcaccctgc
     3721 ccatgagctg cgcccaggag agcggcatgg atagacaccc tgctgcttgc gccagcgcca
     3781 ggatcaacgt cttgaagttc ctattctcta gaaagtatag gaacttctag tgaggaagtg
     3841 aacgtagacc acaagtgctg ttgcctgtaa tcctacccca ggcacaaggg atcttgtcct
     3901 gattcctgtc catcaaaatt gggtgtcctt accatttccg tgcccctaca gtgagagaca
     3961 agatgtccat ccatggtctc tactgcattt tcccttccct tgcagattgt gagatgtaat
     4021 gaaaagtatt gctatttccc agaaataaaa actagattcc tcctgtgttt catacgtttt
     4081 gtttttgtta tgactgaagg acataaaaga ataggatttg gatttggaga cccactttcc
     4141 agaatcttgt cctggcttct cttcatgtgg atcacaatac ggaaagtatg agccaaataa
     4201 aaccattcct ccccaggggg cttttgtcag tgtttgtcat agcaagagag acacaactta
     4261 aggtgccaga ttcgtaagct attgctatga tagactgacc ttgttttgca aaggattttg
     4321 gaagtgtttg taactttggt ttggaaatta agagtgttga gaaatgcaga tgataaactt
     4381 aggaagtttc aggggggaac aaagactggg taattttgta ctatatttga attaagaatg
     4441 tgtggtttct gcacagctgg ggctgaagaa ttgactgtga ataacaagac accaatacca
     4501 ctgacgtgaa acttgttttt ctacaacagt caatgctggt tacctggggc taaagaatca
     4561 actaattaac agaaggccgg cactattgag gtgaaatcta ggaagtattt cctcagggtc
     4621 agcacacaaa agtagtggtc cagagagggc caagactgca tcttgggcct ggtctattta
     4681 aaaagtctcc agtaagcagt ggtcctctgg taatccttca gttcctgcct cctggttcca
     4741 gccctgagcg cctgccctgg tatctatcac tcagtgatag gctgtaatat ggaagtgtaa
     4801 tccaaataaa ccctttcctc cccagggtgc gagctccagc ttttgttccc tttagtgagg
     4861 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4921 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4981 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5041 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5101 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5161 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5221 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5281 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5341 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5401 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5461 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5521 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5581 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5641 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5701 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     5761 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5821 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5881 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5941 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6001 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6061 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6121 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6181 gataccgcgg ctgccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6241 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6301 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6361 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6421 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6481 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6541 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6601 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6661 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6721 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6781 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6841 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6901 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6961 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7021 tccccgaaaa gtgccac
//
