LOCUS       sequence-233463-        7136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Rad21 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-Rad21-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"
     CDS             1706..1717
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1709..1762
                     /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(1763..1796)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1805..1825
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3788..3907
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3910..3943
                     /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 gaggaagtag tcatttcagc aggcagtttt ttctcaattc tggtggccat
      721 ttattcagga tgaagatgct tcagggggtg atcaggatca agaggaaagg agatggaaca
      781 aacgcactca gcagatgctt catggtcttc aggtatgcca gcacagtcca caaagagtcc
      841 tttcccagaa agcaagtcct gttaaacccc gaagcatgcc ttggtgggaa tttctccagg
      901 gctgcattgt tttaaagcat cttagttaca tacatgaagt gtcaagccag gggtaaacct
      961 agagcattta gtttatgtag ccatgattat cctaaaaaac ctgatgcatg gtgctgtgat
     1021 ggggagcgca gctgcgtact gtcttgtttg attagcaaaa gccatgtgct aagtcataga
     1081 gtagcagaag tcagcaacct cagctagggt tccagataca ggtgaggctg gagcacccgt
     1141 gacagttctc ccaagttcct tcctttctgc acatggggat ggatgatact tagggttgga
     1201 aggttatcag gggccggttt gattttggtt ttgttttcac tttaaaatct gctgagtgtt
     1261 tgtttttgct aactcacatc tctgttgtgg cgccttcact tacttcttga actctgtatg
     1321 ccttaagata atatgcttta gataataagg cctttacctt agtgactagc atgggaacca
     1381 cttgcgtaac tacacgaatg tccagtccca gctccagcca aacggagctc actttgacca
     1441 gtgccaaaat tgcatcttct ggttactact ttgtgcgtga gttacttgaa atcatctgct
     1501 ttgttttgtt ctatttcagc gagctcttgc taaaactgga gcagagtcta tcagtttgct
     1561 tgagctgtgt cgaaacacaa accgaaagca ggcagcagca aagttctaca gctttttggt
     1621 tcttaagaag cagcaagcca tcgagctcac acaggaagag ccgtacagtg acatcattgc
     1681 aacccctgga ccacggttcc atattatcga gggaagaggt agcctgctaa cttgcgggga
     1741 cgtagaggaa aatcctggtc ccataacttc gtatagcata cattatacga agttatcaat
     1801 gggcccaaaa aagaaaagaa aagttgaatt catggccaga aaagtggacc tgaccagttg
     1861 tgaccgagag ccgatacaca ttcctggctc tatccagcca tgtgggtgtt tgctggcctg
     1921 cgatgcccag gcagtcagga ttactcgaat taccgaaaat gcgggtgcct tctttggccg
     1981 tgaaacgccc cgtgtcggcg aactcttagc cgattatttc ggagaaaccg aggctcacgc
     2041 acttaggaac gcactggcgc aatcaagcga tcccaaacgc cctgctctca tctttgggtg
     2101 gagagatgga ctgacaggcc gtactttcga cataagcctt caccggcatg acggaaccag
     2161 tataattgag tttgaaccgg ccgccgctga gcaagcagac aacccccttc gcctgaccag
     2221 acaaattatc gcccgcacta aggagctgaa atcgctggaa gaaatggccg ctcgtgttcc
     2281 caggtatctt caggcaatgc tgggttatca tagggtaatg ctctacagat tcgccgacga
     2341 tggctctggt atggtaatag gggaggccaa gcgatccgac ctggagtctt ttctggggca
     2401 gcatttcccc gcatctctag taccccagca ggcaaggctt ttgtacctca agaatgccat
     2461 ccgcgtggta tcggactccc gcggtattag tagcaggatc gttccagaac acgacgcctc
     2521 tggagctgcg ctggatctgt ctttcgctca cctccgatct atcagccctt gtcatttaga
     2581 gtttctgcga aatatggggg tcagcgcaag tatgtccctg agcataataa tcgatggaac
     2641 actatggggc ctaataattt gccatcatta cgaacctaga gcagtaccaa tggcacagcg
     2701 agtggccgcc gaaatgttcg ccgatttttt gtctcttcat tttactgccg ctcaccatca
     2761 gcgcggccac gggactggct cgacaggttc tggcagtagt gggactgctt catcagaaga
     2821 taataacatg gccatggcca gaaaagtgga cctgaccagt tgtgaccgag agccgataca
     2881 cattcctggc tctatccagc catgtgggtg tttgctggcc tgcgatgccc aggcagtcag
     2941 gattactcga attaccgaaa atgcgggtgc cttctttggc cgtgaaacgc cccgtgtcgg
     3001 cgaactctta gccgattatt tcggagaaac cgaggctcac gcacttagga acgcactggc
     3061 gcaatcaagc gatcccaaac gccctgctct catctttggg tggagagatg gactgacagg
     3121 ccgtactttc gacataagcc ttcaccggca tgacggaacc agtataattg agtttgaacc
     3181 ggccgccgct gagcaagcag acaaccccct tcgcctgacc agacaaatta tcgcccgcac
     3241 taaggagctg aaatcgctgg aagaaatggc cgctcgtgtt cccaggtatc ttcaggcaat
     3301 gctgggttat catagggtaa tgctctacag attcgccgac gatggctctg gtatggtaat
     3361 aggggaggcc aagcgatccg acctggagtc ttttctgggg cagcatttcc ccgcatctct
     3421 agtaccccag caggcaaggc ttttgtacct caagaatgcc atccgcgtgg tatcggactc
     3481 ccgcggtatt agtagcagga tcgttccaga acacgacgcc tctggagctg cgctggatct
     3541 gtctttcgct cacctccgat ctatcagccc ttgtcattta gagtttctgc gaaatatggg
     3601 ggtcagcgca agtatgtccc tgagcataat aatcgatgga acactatggg gcctaataat
     3661 ttgccatcat tacgaaccta gagcagtacc aatggcacag cgagtggccg ccgaaatgtt
     3721 cgccgatttt ttgtctcttc attttactgc cgctcaccat cagcgcggat ccgcgtttaa
     3781 actcgagtct cacggcttcc ctcccgaggt ggaggagcag gccgccggca ccctgcccat
     3841 gagctgcgcc caggagagcg gcatggatag acaccctgct gcttgcgcca gcgccaggat
     3901 caacgtcttg aagttcctat tctctagaaa gtataggaac ttctagtgag gagctagatg
     3961 tgttcgagct agtgataact cactagtaca tacaaattgc ccccgtgtgc agggcaccaa
     4021 aaccctttaa gaaagttttt agatttctgt ttgtacaaaa atctttgcct tttctttctt
     4081 ctttttcccc ccagtgtttc taattttgtc aaccatattt ttaagggaaa ctgcttattt
     4141 gggttgggtt tgtattcctg gagaaaacag tagcccaaga acccagaaga cttttaacag
     4201 ttcagaacag atgtgtgcaa tattggtgca tgtaagaata tggagtaaca gtcaaaaggc
     4261 accattttta atgttagttt tccattacta tgttgaaagg aaaacctgcc taggaaaatg
     4321 cctgacactt taagaactgt ggtttgagtc ccttgacagg aagagaaaaa tgtcttccca
     4381 tcagtgaaac caacggtctg gttaaccact gtagtaggga tagtgtgtga agcatcgtgc
     4441 ttgtcttctt gatcatgttt aaagtaaatc cacactgagc aagaatttgt gaggttgtgc
     4501 tgactgatct tcttcggcta ttatgttctg aaatcattcc gtgagtctta ggacctctga
     4561 tgctatgcac cgcagctcag aacgatcaga cgtctacagt ggcgtgactc ctcaggggct
     4621 gcagatcact ctcagaggtg tttggatgtt tgcttgaata atgagatcat ggaagcagac
     4681 attcccctgc ctgctgaagg gcagaccaat gagagactac cccagattga cttcctttaa
     4741 gcaaacagtg ctgtaaaaac taatggcttc tctgatattt attataaatg ttagtactca
     4801 tctttttcca aggctgcaca ctcctgtatt tgcaacttat ttttaataac tttgcaacta
     4861 taatcctgta tcagtttcct ataatttaag tggagaaaaa catcctaata agcggccgcc
     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
//
