LOCUS       sequence-238854-        7087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Klf4 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 7087)
  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 7087)
  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-Klf4-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7087
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        37..127
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(128..196,197..988)
                     /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     complement(346..365)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1159..1747
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1648..1667
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1901..1918
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2035..2056
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2071..2076,2077..2094,2095..2101)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2109..2125
                     /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)."
     primer_bind     2114..2136
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2133..2149
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2133..2149
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2168..2188
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2170..2188
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             3242..3295
                     /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(3296..3329)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3338..3358
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5321..5440
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    5443..5476
                     /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(6443..6462)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6444..6462)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6472..6489)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6472..6488)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6481..6503)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6630..7085
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6717..6736)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6927..6948
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa tccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaag
      121 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
      181 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
      241 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
      301 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
      361 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
      421 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
      481 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
      541 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
      601 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
      661 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
      721 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
      781 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
      841 tggcagccgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
      901 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
      961 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     1021 gattgattta aaacttcatt tttaatttag aaggatctag gtgaagatcc tttttgataa
     1081 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1141 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1201 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1261 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     1321 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1381 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1441 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1501 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     1561 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     1621 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     1681 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     1741 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     1801 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     1861 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     1921 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     1981 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     2041 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     2101 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     2161 caagcgcgca attaaccctc actaaaggga acaaaagctg cgtacgtctg gtcaagaacc
     2221 cgagtctcga aataacaaag aacgcacagg tagtctttta gtgggtagag tcttccagat
     2281 gcatttatga aaacagccag aacggctctg ctggagaaat tagccacggg tggcattctg
     2341 gacccagcgt aatggcatgg gatcctttgg gtttcaacag agccaacttc tttttttttt
     2401 tttccatttt ttattaagta tttagctcat ttacatttcc aatgctatac caaaagtccc
     2461 ccatacccac ccacccccac tcccctaccc gcccactccc cctttttggc cctggcgttc
     2521 ccctgtactg gggcatataa agtttgcctg tccaatgggc ctctctttcc agtgatgacc
     2581 gactaggcca tcttttgata tatatgcaac tagagacaag agctccaggg tactggttag
     2641 ttcataatgt tgttccacct ataggattgc agatcccttt agcagagcca acttctaatc
     2701 cagtccattt ccagtgctgg ttaggctcga gagtgaaaac tccgtactgt gggggtggac
     2761 gctttgagag ggtatctgct tttttttttt taagttacta actactgttt ctccatgagt
     2821 ttaaactcat aacacatgtc attcctcttc atctgaaatt cagtatggcc ctaaagtgtc
     2881 ataaagacat tcctgaaggt tttttttatt attattatta tatttttact ttcttgaaag
     2941 gagagagggt ggggcatgca gtagaatttg ctctctggcc tctccctttt ttttaagaaa
     3001 aaaaaggggg gtggctaatt cttccttcta gcaatggctt cagcagcccg ttctctttcc
     3061 ctttacaggc gagaaacctt accactgtga ctgggacggc tgtgggtgga aattcgcccg
     3121 ctccgatgaa ctgaccaggc actaccgcaa acacacaggg caccggccct ttcagtgcca
     3181 gaagtgtgac agggcctttt ccaggtcgga ccaccttgcc ttacacatga agaggcactt
     3241 tgagggaaga ggtagcctgc taacttgcgg ggacgtagag gaaaatcctg gtcccataac
     3301 ttcgtatagc atacattata cgaagttatc aatgggccca aaaaagaaaa gaaaagttga
     3361 attcatggcc agaaaagtgg acctgaccag ttgtgaccga gagccgatac acattcctgg
     3421 ctctatccag ccatgtgggt gtttgctggc ctgcgatgcc caggcagtca ggattactcg
     3481 aattaccgaa aatgcgggtg ccttctttgg ccgtgaaacg ccccgtgtcg gcgaactctt
     3541 agccgattat ttcggagaaa ccgaggctca cgcacttagg aacgcactgg cgcaatcaag
     3601 cgatcccaaa cgccctgctc tcatctttgg gtggagagat ggactgacag gccgtacttt
     3661 cgacataagc cttcaccggc atgacggaac cagtataatt gagtttgaac cggccgccgc
     3721 tgagcaagca gacaaccccc ttcgcctgac cagacaaatt atcgcccgca ctaaggagct
     3781 gaaatcgctg gaagaaatgg ccgctcgtgt tcccaggtat cttcaggcaa tgctgggtta
     3841 tcatagggta atgctctaca gattcgccga cgatggctct ggtatggtaa taggggaggc
     3901 caagcgatcc gacctggagt cttttctggg gcagcatttc cccgcatctc tagtacccca
     3961 gcaggcaagg cttttgtacc tcaagaatgc catccgcgtg gtatcggact cccgcggtat
     4021 tagtagcagg atcgttccag aacacgacgc ctctggagct gcgctggatc tgtctttcgc
     4081 tcacctccga tctatcagcc cttgtcattt agagtttctg cgaaatatgg gggtcagcgc
     4141 aagtatgtcc ctgagcataa taatcgatgg aacactatgg ggcctaataa tttgccatca
     4201 ttacgaacct agagcagtac caatggcaca gcgagtggcc gccgaaatgt tcgccgattt
     4261 tttgtctctt cattttactg ccgctcacca tcagcgcggc cacgggactg gctcgacagg
     4321 ttctggcagt agtgggactg cttcatcaga aaataataac atggccatgg ccagaaaagt
     4381 ggacctgacc agttgtgacc gagagccgat acacattcct ggctctatcc agccatgtgg
     4441 gtgtttgctg gcctgcgatg cccaggcagt caggattact cgaattaccg aaaatgcggg
     4501 tgccttcttt ggccgtgaaa cgccccgtgt cggcgaactc ttagccgatt atttcggaga
     4561 aaccgaggct cacgcactta ggaacgcact ggcgcaatca agcgatccca aacgccctgc
     4621 tctcatcttt gggtggagag atggactgac aggccgtact ttcgacataa gccttcaccg
     4681 gcatgacgga accagtataa ttgagtttga accggccgcc gctgagcaag cagacaaccc
     4741 ccttcgcctg accagacaaa ttatcgcccg cactaaggag ctgaaatcgc tggaagaaat
     4801 ggccgctcgt gttcccaggt atcttcaggc aatgctgggt tatcataggg taatgctcta
     4861 cagattcgcc gacgatggct ctggtatggt aataggggag gccaagcgat ccgacctgga
     4921 gtcttttctg gggcagcatt tccccgcatc tctagtaccc cagcaggcaa ggcttttgta
     4981 cctcaagaat gccatccgcg tggtatcgga ctcccgcggt attagtagca ggatcgttcc
     5041 agaacacgac gcctctggag ctgcgctgga tctgtctttc gctcacctcc gatctatcag
     5101 cccttgtcat ttagagtttc tgcgaaatat gggggtcagc gcaagtatgt ccctgagcat
     5161 aataatcgat ggaacactat ggggcctaat aatttgccat cattacgaac ctagagcagt
     5221 accaatggca cagcgagtgg ccgccgaaat gttcgccgat tttttgtctc ttcattttac
     5281 tgccgctcac catcagcgcg gatccgcgtt taaactcgag tctcacggct tccctcccga
     5341 ggtggaggag caggccgccg gcaccctgcc catgagctgc gcccaggaga gcggcatgga
     5401 tagacaccct gctgcttgcg ccagcgccag gatcaacgtc ttgaagttcc tattctctag
     5461 aaagtatagg aacttctaga tcccacgtag tggatgtgac ccacactgcc aggagagaga
     5521 gttcagtatt tttttttcta acctttcaca ctgtcttccc acgaggggag gagcccagct
     5581 ggcaagcgct acaatcatgg tcaagttccc agcaagtcag cttgtgaatg gataatcagg
     5641 agaaaggaag agttcaagag acaaaacaga aatactaaaa acaaacaaac aaaaaaacaa
     5701 acaaaaaaaa caagaaaaaa aaatcacaga acagatgggg tctgatactg gatggatctt
     5761 ctatcattcc aataccaaat ccaacttgaa catgcccgga cttacaaaat gccaaggggt
     5821 gactggaagt ttgtggatat cagggtatac actaaatcag tgagcttggg gggagggaag
     5881 accaggattc ccttgaatcg tgtttcgatg atgcaataca cacgtaaaga tcaccttgta
     5941 tgctctttgc cttcttaaaa aaaaaaaaag ccattattgt gtcggaggaa gaggaagcga
     6001 ttcaggtaca gaacatgttc taacagccta aatgatggtg cttggtgagt cgtggttcta
     6061 aaggtaccaa acgggggagc caaagttctc caactgctgc atacttttga caaggaaaat
     6121 ctagttttgt cttccgatct acattgatga cctaagccag gtaaataagc ctggtttatt
     6181 tctgtaacat ttttatgcag acagtctgtt atgcactgtg gtttcagatg tgcaataatt
     6241 tgtacaatgg tttattccca agtatgcctt taagcagaac aaatgtgttt ttctatatag
     6301 ttccttgcct taataaatat gtaatataaa tttaagcaaa cttctatttt gtatatttgt
     6361 aaactacaaa gtaaaaaaaa atgaacattt tgtggagttt gtattttgca tactcaaggt
     6421 gagaaataag aagctcaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt
     6481 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     6541 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     6601 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     6661 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     6721 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     6781 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     6841 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     6901 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     6961 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     7021 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     7081 aatttag
//
