LOCUS       sequence-238828-        7249 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 7249)
  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 7249)
  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..7249
                     /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             1635..1646
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1638..1691
                     /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(1692..1725)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1734..1754
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3717..3836
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3839..3872
                     /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(5061..5081)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5061..5079)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5100..5116)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5100..5116)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5113..5135)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5124..5140
                     /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(5148..5154,5155..5172,5173..5178))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5193..5214
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5331..5348)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5502..6090)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5582..5601)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6261..7052,7053..7121))
                     /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     6884..6903
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7122..7226)
                     /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 gtgtgtggct
      901 gacctatggc tgttgggtat ggatcccaga tttcctgtaa gagcagtcag tggctctgcc
      961 tttgcttccc agtgtcatcc tgatccatct atgtccatta gtcctgtccc tttcccatcc
     1021 ctcttcctcc acgcactctt tcttgtgatc tttggttctt tgaggcaggc tctctctatg
     1081 tagccctggc agtcagagat ctgcctgcct agtctcagga gtgctggggt taacggcctg
     1141 tgcggccatg ctaatctttt cagtatttct gcagtttaca tctttagagt ccccttttca
     1201 ctcattgcat taatcacctc tttcagaagg gctcagcacc agtggagtat cccagcatcc
     1261 attgcagcta tccccagggc tatctggtga acgcatctgg aagcctttcc atgtggggca
     1321 gccagacttg gaccaaccca acttggagca gccagacctg gaccaaccca acttggaaca
     1381 accagacctg gaccaaccca acttggagca gccaggcctg gaccgctcag tcctggaacg
     1441 gccagccttg gaatgctgct ccgctccata acttcgggga ggactttctg cagccttacg
     1501 tacagttgca gcaaaacttc tctgccagtg atttggaggt gaatttggaa gccactaggg
     1561 aaagccatgc gcattttagc accccacaag ccttggaatt attcctgaac tactctgtga
     1621 ctccaccagg tgaaatagag ggaagaggta gcctgctaac ttgcggggac gtagaggaaa
     1681 atcctggtcc cataacttcg tatagcatac attatacgaa gttatcaatg ggcccaaaaa
     1741 agaaaagaaa agttgaattc atggccagaa aagtggacct gaccagttgt gaccgagagc
     1801 cgatacacat tcctggctct atccagccat gtgggtgttt gctggcctgc gatgcccagg
     1861 cagtcaggat tactcgaatt accgaaaatg cgggtgcctt ctttggccgt gaaacgcccc
     1921 gtgtcggcga actcttagcc gattatttcg gagaaaccga ggctcacgca cttaggaacg
     1981 cactggcgca atcaagcgat cccaaacgcc ctgctctcat ctttgggtgg agagatggac
     2041 tgacaggccg tactttcgac ataagccttc accggcatga cggaaccagt ataattgagt
     2101 ttgaaccggc cgccgctgag caagcagaca acccccttcg cctgaccaga caaattatcg
     2161 cccgcactaa ggagctgaaa tcgctggaag aaatggccgc tcgtgttccc aggtatcttc
     2221 aggcaatgct gggttatcat agggtaatgc tctacagatt cgccgacgat ggctctggta
     2281 tggtaatagg ggaggccaag cgatccgacc tggagtcttt tctggggcag catttccccg
     2341 catctctagt accccagcag gcaaggcttt tgtacctcaa gaatgccatc cgcgtggtat
     2401 cggactcccg cggtattagt agcaggatcg ttccagaaca cgacgcctct ggagctgcgc
     2461 tggatctgtc tttcgctcac ctccgatcta tcagcccttg tcatttagag tttctgcgaa
     2521 atatgggggt cagcgcaagt atgtccctga gcataataat cgatggaaca ctatggggcc
     2581 taataatttg ccatcattac gaacctagag cagtaccaat ggcacagcga gtggccgccg
     2641 aaatgttcgc cgattttttg tctcttcatt ttactgccgc tcaccatcag cgcggccacg
     2701 ggactggctc gacaggttct ggcagtagtg ggactgcttc atcagaaaat aataacatgg
     2761 ccatggccag aaaagtggac ctgaccagtt gtgaccgaga gccgatacac attcctggct
     2821 ctatccagcc atgtgggtgt ttgctggcct gcgatgccca ggcagtcagg attactcgaa
     2881 ttaccgaaaa tgcgggtgcc ttctttggcc gtgaaacgcc ccgtgtcggc gaactcttag
     2941 ccgattattt cggagaaacc gaggctcacg cacttaggaa cgcactggcg caatcaagcg
     3001 atcccaaacg ccctgctctc atctttgggt ggagagatgg actgacaggc cgtactttcg
     3061 acataagcct tcaccggcat gacggaacca gtataattga gtttgaaccg gccgccgctg
     3121 agcaagcaga caaccccctt cgcctgacca gacaaattat cgcccgcact aaggagctga
     3181 aatcgctgga agaaatggcc gctcgtgttc ccaggtatct tcaggcaatg ctgggttatc
     3241 atagggtaat gctctacaga ttcgccgacg atggctctgg tatggtaata ggggaggcca
     3301 agcgatccga cctggagtct tttctggggc agcatttccc cgcatctcta gtaccccagc
     3361 aggcaaggct tttgtacctc aagaatgcca tccgcgtggt atcggactcc cgcggtatta
     3421 gtagcaggat cgttccagaa cacgacgcct ctggagctgc gctggatctg tctttcgctc
     3481 acctccgatc tatcagccct tgtcatttag agtttctgcg aaatatgggg gtcagcgcaa
     3541 gtatgtccct gagcataata atcgatggaa cactatgggg cctaataatt tgccatcatt
     3601 acgaacctag agcagtacca atggcacagc gagtggccgc cgaaatgttc gccgattttt
     3661 tgtctcttca ttttactgcc gctcaccatc agcgcggatc cgcgtttaaa ctcgagtctc
     3721 acggcttccc tcccgaggtg gaggagcagg ccgccggcac cctgcccatg agctgcgccc
     3781 aggagagcgg catggataga caccctgctg cttgcgccag cgccaggatc aacgtcttga
     3841 agttcctatt ctctagaaag tataggaact tctaggactt acgcaacatc tgggcttaaa
     3901 gtcagggcaa agccaggttc cttccttctt ccaaatattt tcatattttt tttaaagatt
     3961 tatttattca ttatatgtaa gtacactgta gctgtcttca gacactccag aagagggcgt
     4021 cagatcttgt tacgtatggt tgtgagccac catgtggttg ctgggatttg aactcctgac
     4081 cttcggaaga gcagtcgggt gctcttatcc actgagccat ctcaccagcc cctggtttat
     4141 ttttttaatt attatttgct ttttgtttat caagacaggg tttctctgca tagctctaat
     4201 tgtctttgaa ctagctctgc agaccagcct ggccttgaac tcagagatct gcccacttat
     4261 ctttgcctcc tgaatgctgg gaccaaaggt ggcataccac cacacctggc atatatattg
     4321 tttatttcta tttctatttt tattggtgcc agagcaaacc taggacttag aacatgctgg
     4381 gcaccaactc aacttctgag ctctatttac aacttggtgt gttagtgtat ttgtcttagt
     4441 tctgaatttg tccttttttt agtgttaact ctaggctttg gagacagtga ggtgcatata
     4501 ctctctcctt cccaagaata agtgcttgaa cacccttacc cacgcccacc cacccatgct
     4561 agtctttttt cttagaagcg tgggtcttgg tatacactgt gtcattttga ggggtgaggt
     4621 ttaaaagtat atacaaagta taacgatatg gtggctactc tcgaggatga gacagaagga
     4681 ccaggagttt gagggtagct cagatatgca ataagttcaa ggccaacctg tactatgttt
     4741 aaatagtaag acagcatctc gataaaataa taaaactaaa gtctcaacaa aataaaagct
     4801 ttcacctatt aaggtgcttg cttgtccttg gagtccccca agagtaactg ctatgttaat
     4861 atctgtagaa agatgtttat atttgactgt accatgatga accgatgcca gctggactag
     4921 tttaaacaaa ataaaacact aattttacct ttagtggtct tatcttttgt ccagtggggt
     4981 caatatactt tttattttgg tatgtgaagg ggagggtctt aaccgaggcc gccaccgcgg
     5041 tggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     5101 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5161 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5221 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagttgca ttaatgaatc
     5281 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5341 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5401 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5461 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5521 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5581 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5641 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5701 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5761 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5821 ccggtaagac acgacttatc accactggca gcagccactg gtaacaggat tagcagagcg
     5881 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5941 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6001 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6061 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6121 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6181 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6241 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6301 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6361 gagggcttac catctggccc cagtgctgca atgataccgc ggctgccacg ctcaccggct
     6421 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6481 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6541 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6601 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6661 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6721 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6781 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6841 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6901 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6961 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7021 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7081 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7141 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7201 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
