LOCUS       sequence-233470-        7253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Nanog 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 7253)
  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 7253)
  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-mNanog-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7253
                     /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             1639..1650
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1642..1695
                     /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(1696..1729)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1738..1758
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3721..3840
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3843..3876
                     /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(5065..5085)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5065..5083)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5104..5120)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5104..5120)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5117..5139)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5128..5144
                     /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(5152..5158,5159..5176,5177..5182))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5197..5218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5335..5352)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5506..6094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5586..5605)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6265..7056,7057..7125))
                     /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     6888..6907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7126..7230)
                     /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 gacattctgt atgtcttagc ttgttcgaat gtcctgtcac tctgcaggtt
      721 aagacctggt ttcaaaacca aaggatgaag tgcaagcggt ggcagaaaaa ccagtggttg
      781 aagactagca atggtctgat tcaggtaaca agatgttttc tctgctttcc tttttccccc
      841 tccatcttac catcttattt tatgtgtctg tgccgtgtgt gtgtgtgtgt gtgtgtgtgt
      901 ggctgaccta tggctgttgg gtatggatcc cagatttcct gtaagagcag tcagtggctc
      961 tgcctttgct tcccagtgtc atcctgatcc atctatgtcc attagtcctg tccctttccc
     1021 atccctcttc ctccacgcac tctttcttgt gatctttggt tctttgaggc aggctctctc
     1081 tatgtagccc tggcagtcag agatctgcct gcctagtctc aggagtgctg gggttaacgg
     1141 cctgtgcggc catgctaatc ttttcagtat ttctgcagtt tacatcttta gagtcccctt
     1201 ttcactcatt gcattaatca cctctttcag aagggctcag caccagtgga gtatcccagc
     1261 atccattgca gctatcccca gggctatctg gtgaacgcat ctggaagcct ttccatgtgg
     1321 ggcagccaga cttggaccaa cccaacttgg agcagccaga cctggaccaa cccaacttgg
     1381 aacaaccaga cctggaccaa cccaacttgg agcagccagg cctggaccgc tcagtcctgg
     1441 aacggccagc cttggaatgc tgctccgctc cataacttcg gggaggactt tctgcagcct
     1501 tacgtacagt tgcagcaaaa cttctctgcc agtgatttgg aggtgaattt ggaagccact
     1561 agggaaagcc atgcgcattt tagcacccca caagccttgg aattattcct gaactactct
     1621 gtgactccac caggtgaaat agagggaaga ggtagcctgc taacttgcgg ggacgtagag
     1681 gaaaatcctg gtcccataac ttcgtatagc atacattata cgaagttatc aatgggccca
     1741 aaaaagaaaa gaaaagttga attcatggcc agaaaagtgg acctgaccag ttgtgaccga
     1801 gagccgatac acattcctgg ctctatccag ccatgtgggt gtttgctggc ctgcgatgcc
     1861 caggcagtca ggattactcg aattaccgaa aatgcgggtg ccttctttgg ccgtgaaacg
     1921 ccccgtgtcg gcgaactctt agccgattat ttcggagaaa ccgaggctca cgcacttagg
     1981 aacgcactgg cgcaatcaag cgatcccaaa cgccctgctc tcatctttgg gtggagagat
     2041 ggactgacag gccgtacttt cgacataagc cttcaccggc atgacggaac cagtataatt
     2101 gagtttgaac cggccgccgc tgagcaagca gacaaccccc ttcgcctgac cagacaaatt
     2161 atcgcccgca ctaaggagct gaaatcgctg gaagaaatgg ccgctcgtgt tcccaggtat
     2221 cttcaggcaa tgctgggtta tcatagggta atgctctaca gattcgccga cgatggctct
     2281 ggtatggtaa taggggaggc caagcgatcc gacctggagt cttttctggg gcagcatttc
     2341 cccgcatctc tagtacccca gcaggcaagg cttttgtacc tcaagaatgc catccgcgtg
     2401 gtatcggact cccgcggtat tagtagcagg atcgttccag aacacgacgc ctctggagct
     2461 gcgctggatc tgtctttcgc tcacctccga tctatcagcc cttgtcattt agagtttctg
     2521 cgaaatatgg gggtcagcgc aagtatgtcc ctgagcataa taatcgatgg aacactatgg
     2581 ggcctaataa tttgccatca ttacgaacct agagcagtac caatggcaca gcgagtggcc
     2641 gccgaaatgt tcgccgattt tttgtctctt cattttactg ccgctcacca tcagcgcggc
     2701 cacgggactg gctcgacagg ttctggcagt agtgggactg cttcatcaga agataataac
     2761 atggccatgg ccagaaaagt ggacctgacc agttgtgacc gagagccgat acacattcct
     2821 ggctctatcc agccatgtgg gtgtttgctg gcctgcgatg cccaggcagt caggattact
     2881 cgaattaccg aaaatgcggg tgccttcttt ggccgtgaaa cgccccgtgt cggcgaactc
     2941 ttagccgatt atttcggaga aaccgaggct cacgcactta ggaacgcact ggcgcaatca
     3001 agcgatccca aacgccctgc tctcatcttt gggtggagag atggactgac aggccgtact
     3061 ttcgacataa gccttcaccg gcatgacgga accagtataa ttgagtttga accggccgcc
     3121 gctgagcaag cagacaaccc ccttcgcctg accagacaaa ttatcgcccg cactaaggag
     3181 ctgaaatcgc tggaagaaat ggccgctcgt gttcccaggt atcttcaggc aatgctgggt
     3241 tatcataggg taatgctcta cagattcgcc gacgatggct ctggtatggt aataggggag
     3301 gccaagcgat ccgacctgga gtcttttctg gggcagcatt tccccgcatc tctagtaccc
     3361 cagcaggcaa ggcttttgta cctcaagaat gccatccgcg tggtatcgga ctcccgcggt
     3421 attagtagca ggatcgttcc agaacacgac gcctctggag ctgcgctgga tctgtctttc
     3481 gctcacctcc gatctatcag cccttgtcat ttagagtttc tgcgaaatat gggggtcagc
     3541 gcaagtatgt ccctgagcat aataatcgat ggaacactat ggggcctaat aatttgccat
     3601 cattacgaac ctagagcagt accaatggca cagcgagtgg ccgccgaaat gttcgccgat
     3661 tttttgtctc ttcattttac tgccgctcac catcagcgcg gatccgcgtt taaactcgag
     3721 tctcacggct tccctcccga ggtggaggag caggccgccg gcaccctgcc catgagctgc
     3781 gcccaggaga gcggcatgga tagacaccct gctgcttgcg ccagcgccag gatcaacgtc
     3841 ttgaagttcc tattctctag aaagtatagg aacttctagg acttacgcaa catctgggct
     3901 taaagtcagg gcaaagccag gttccttcct tcttccaaat attttcatat tttttttaaa
     3961 gatttattta ttcattatat gtaagtacac tgtagctgtc ttcagacact ccagaagagg
     4021 gcgtcagatc ttgttacgta tggttgtgag ccaccatgtg gttgctggga tttgaactcc
     4081 tgaccttcgg aagagcagtc gggtgctctt atccactgag ccatctcacc agcccctggt
     4141 ttattttttt aattattatt tgctttttgt ttatcaagac agggtttctc tgcatagctc
     4201 taattgtctt tgaactagct ctgcagacca gcctggcctt gaactcagag atctgcccac
     4261 ttatctttgc ctcctgaatg ctgggaccaa aggtggcata ccaccacacc tggcatatat
     4321 attgtttatt tctatttcta tttttattgg tgccagagca aacctaggac ttagaacatg
     4381 ctgggcacca actcaacttc tgagctctat ttacaacttg gtgtgttagt gtatttgtct
     4441 tagttctgaa tttgtccttt ttttagtgtt aactctaggc tttggagaca gtgaggtgca
     4501 tatactctct ccttcccaag aataagtgct tgaacaccct tacccacgcc cacccaccca
     4561 tgctagtctt ttttcttaga agcgtgggtc ttggtataca ctgtgtcatt ttgaggggtg
     4621 aggtttaaaa gtatatacaa agtataacga tatggtggct actctcgagg atgagacaga
     4681 aggaccagga gtttgagggt agctcagata tgcaataagt tcaaggccaa cctgtactat
     4741 gtttaaatag taagacagca tctcgataaa ataataaaac taaagtctca acaaaataaa
     4801 agctttcacc tattaaggtg cttgcttgtc cttggagtcc cccaagagta actgctatgt
     4861 taatatctgt agaaagatgt ttatatttga ctgtaccatg atgaaccgat gccagctgga
     4921 ctagtttaaa caaaataaaa cactaatttt acctttagtg gtcttatctt ttgtccagtg
     4981 gggtcaatat actttttatt ttggtatgtg aaggggaggg tcttaaccga ggccgccacc
     5041 gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     5101 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5161 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5221 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5281 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5341 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5401 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5461 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5521 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5581 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5641 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5701 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5761 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5821 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5881 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5941 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6001 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6061 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6121 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6181 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6241 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6301 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6361 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcggctgc cacgctcacc
     6421 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6481 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6541 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6601 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6661 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6721 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6781 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6841 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6901 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6961 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7021 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7081 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7141 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7201 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cac
//
