LOCUS       sequence-238126-        7135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Cdt1 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 7135)
  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 7135)
  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-Cdt1-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7135
                     /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             1672..1725
                     /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(1726..1759)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1768..1788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3751..3870
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3873..3906
                     /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..7099)
                     /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 gagttatgaa tcttttgtga cctctgacct gtgcctaccc tgcagatgga
      721 gaaggccctg agcaacctgg ccctgcgctc ggctgagccc ggtagccctg ggacctctac
      781 tccaccactc ccggccactc cgccagccac cccacctgct gcctctccga gcgccctgaa
      841 gggtgtgtcc caagcactgc tagagcgggt gagtcctggg tctcgggtag ggtggacttg
      901 gtgacaggag tgggctttcc ttgttgacca ttggctgctg acccatcccc atgcagataa
      961 gggccaagga ggtccagaag cagctggcaa ggatgacacg gtgccccgag caggagcttc
     1021 gcctgcagcg gttagagcgt ctgccagagc tggcccgcgt gctgcgcaat gtcttcgtgt
     1081 ctgagcggaa gccggcactc actatggagg tggtctgtgc aaggatggtg gacagttgcc
     1141 aaactgctct gagtccaggt ttgtgcaaag caaaacaagg gtgagagggt gggcatggtc
     1201 atgctcactg tgttcttggc aaggagctag gtctatttct gaccagggac agtttgtctg
     1261 ccatggtctt ggggaccccc aattctgcca cttataggcc tgctcaccag gctcggagca
     1321 cgggtgtctt tgcctctttg ctgttcgttc tcctggtgct tgagcacggt gtcccccatg
     1381 gctcttggct ctgcttggat ccactgtaca tagcagtgga taagccttga caaaggccta
     1441 ttaatgggat gtcgggcacc tgggtgcgtg agagcaaatc atctaggcct acacccacaa
     1501 tccacctgtg caggggagat ggagaaacat ctggtgctcc tggcagagtt gctgccggac
     1561 tggctcagcc tgcatcgcat ccgcacggat acctacgtca agctggacaa ggctgttgac
     1621 ctggctggcc tcactgcgag gctggcccac cacgtccacg ccgaggggct ggagggaaga
     1681 ggtagcctgc taacttgcgg ggacgtagag gaaaatcctg gtcccataac ttcgtatagc
     1741 atacattata cgaagttatc aatgggccca aaaaagaaaa gaaaagttga attcatggcc
     1801 agaaaagtgg acctgaccag ttgtgaccga gagccgatac acattcctgg ctctatccag
     1861 ccatgtgggt gtttgctggc ctgcgatgcc caggcagtca ggattactcg aattaccgaa
     1921 aatgcgggtg ccttctttgg ccgtgaaacg ccccgtgtcg gcgaactctt agccgattat
     1981 ttcggagaaa ccgaggctca cgcacttagg aacgcactgg cgcaatcaag cgatcccaaa
     2041 cgccctgctc tcatctttgg gtggagagat ggactgacag gccgtacttt cgacataagc
     2101 cttcaccggc atgacggaac cagtataatt gagtttgaac cggccgccgc tgagcaagca
     2161 gacaaccccc ttcgcctgac cagacaaatt atcgcccgca ctaaggagct gaaatcgctg
     2221 gaagaaatgg ccgctcgtgt tcccaggtat cttcaggcaa tgctgggtta tcatagggta
     2281 atgctctaca gattcgccga cgatggctct ggtatggtaa taggggaggc caagcgatcc
     2341 gacctggagt cttttctggg gcagcatttc cccgcatctc tagtacccca gcaggcaagg
     2401 cttttgtacc tcaagaatgc catccgcgtg gtatcggact cccgcggtat tagtagcagg
     2461 atcgttccag aacacgacgc ctctggagct gcgctggatc tgtctttcgc tcacctccga
     2521 tctatcagcc cttgtcattt agagtttctg cgaaatatgg gggtcagcgc aagtatgtcc
     2581 ctgagcataa taatcgatgg aacactatgg ggcctaataa tttgccatca ttacgaacct
     2641 agagcagtac caatggcaca gcgagtggcc gccgaaatgt tcgccgattt tttgtctctt
     2701 cattttactg ccgctcacca tcagcgcggc cacgggactg gctcgacagg ttctggcagt
     2761 agtgggactg cttcatcaga aaataataac atggccatgg ccagaaaagt ggacctgacc
     2821 agttgtgacc gagagccgat acacattcct ggctctatcc agccatgtgg gtgtttgctg
     2881 gcctgcgatg cccaggcagt caggattact cgaattaccg aaaatgcggg tgccttcttt
     2941 ggccgtgaaa cgccccgtgt cggcgaactc ttagccgatt atttcggaga aaccgaggct
     3001 cacgcactta ggaacgcact ggcgcaatca agcgatccca aacgccctgc tctcatcttt
     3061 gggtggagag atggactgac aggccgtact ttcgacataa gccttcaccg gcatgacgga
     3121 accagtataa ttgagtttga accggccgcc gctgagcaag cagacaaccc ccttcgcctg
     3181 accagacaaa ttatcgcccg cactaaggag ctgaaatcgc tggaagaaat ggccgctcgt
     3241 gttcccaggt atcttcaggc aatgctgggt tatcataggg taatgctcta cagattcgcc
     3301 gacgatggct ctggtatggt aataggggag gccaagcgat ccgacctgga gtcttttctg
     3361 gggcagcatt tccccgcatc tctagtaccc cagcaggcaa ggcttttgta cctcaagaat
     3421 gccatccgcg tggtatcgga ctcccgcggt attagtagca ggatcgttcc agaacacgac
     3481 gcctctggag ctgcgctgga tctgtctttc gctcacctcc gatctatcag cccttgtcat
     3541 ttagagtttc tgcgaaatat gggggtcagc gcaagtatgt ccctgagcat aataatcgat
     3601 ggaacactat ggggcctaat aatttgccat cattacgaac ctagagcagt accaatggca
     3661 cagcgagtgg ccgccgaaat gttcgccgat tttttgtctc ttcattttac tgccgctcac
     3721 catcagcgcg gatccgcgtt taaactcgag tctcacggct tccctcccga ggtggaggag
     3781 caggccgccg gcaccctgcc catgagctgc gcccaggaga gcggcatgga tagacaccct
     3841 gctgcttgcg ccagcgccag gatcaacgtc ttgaagttcc tattctctag aaagtatagg
     3901 aacttctagt gactttgagc tccttgcctg tttctttcat cagtacacga cgcacttacg
     3961 cctttaagcc tcgccagtgt gggcagctat cgttgcccat ggatctcata aagtgctggc
     4021 attaagttgc ttcctgaggc tttggggcat cccagactca gctctagggg aagtagactc
     4081 tgaagagtag ggtatgttgg atggccattg cacaaactac tcaagcatta ggtatggtca
     4141 agacccaaga tcaaggtcca ggtggaagcc taggctggca tagccttgct tccccatgag
     4201 acttaagaat cacacagacc ttggactttc ctgatttcac gggacgctgc tctgagagtg
     4261 aaattgggcc ttctgtaaat atgtgaagtg tggtttcttt tcaaacctta tatggccctg
     4321 catgtgactg ctagttttgg cttttaatag agtcatgtaa gatttaaata aaatactact
     4381 gagatgaagc tgaacctgtc accgtaaaat ctctttaaat gctgttgtgt gatgtggggc
     4441 aatgggctat ggacagacat cctggtcctg gtgggtggta attttcacat ggacatggac
     4501 agaaggcgtt ccatcatgtc tcctggaccc ctggctcctg tcgaagttac agcccccaca
     4561 ggagaggcat gtggctttta gtcatgtagc caatgtcaca agcttctggc attctggcta
     4621 gactccttcc cacagttacc tagcaacagc aagatggcaa agcccactat aaggggctgt
     4681 ttggcccctc ctcgctctct taagctctct ggcttttttt tctctttccc ttcccccctc
     4741 tctccacgtg gccatggcct gtccttctac cttctctttc tccttgcctt tctacaataa
     4801 agctctaaaa ccatagactg tctccgatca tgaaggccca ccgtgtttga atgatgatcg
     4861 gataggcttt ctcctaatga gccactttta acctgccaga aggccttcct 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 gtgagacacg acttatcgcc actggcagca gccactggta acaggattag
     5761 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5821 cactagaaga 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 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7081 atttagaaaa ataaacaaat agggattccg cgcacatttc cccgaaaagt gccac
//
