LOCUS       sequence-233467-        7090 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 7090)
  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 7090)
  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..7090
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2169..2189
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             3245..3298
                     /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(3299..3332)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3341..3361
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5324..5443
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    5446..5479
                     /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(6446..6465)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6447..6465)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6475..6492)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6475..6491)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6484..6506)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6633..7088
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6720..6739)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6930..6951
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtggcagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gcgtacgtct ggtcaagaac
     2221 ccgagtctcg aaataacaaa gaacgcacag gtagtctttt agtgggtaga gtcttccaga
     2281 tgcatttatg aaaacagcca gaacggctct gctggagaaa ttagccacgg gtggcattct
     2341 ggacccagcg taatggcatg ggatcctttg ggtttcaaca gagccaactt cttttttttt
     2401 tttttccatt ttttattaag tatttagctc atttacattt ccaatgctat accaaaagtc
     2461 ccccataccc acccaccccc actcccctac ccgcccactc cccctttttg gccctggcgt
     2521 tcccctgtac tggggcatat aaagtttgcc tgtccaatgg gcctctcttt ccagtgatgg
     2581 ccgactaggc catcttttga tatatatgca actagagaca agagctccag ggtactggtt
     2641 agttcataat gttgttccac ctataggatt gcagatccct ttagcagagc caacttctaa
     2701 tccagtccat ttccagtgct ggttaggctc gagagtgaaa actccgtact gtgggggtgg
     2761 acgctttgag agggtatctg cttttttttt ttttaagtta ctaactactg tttctccatg
     2821 agtttaaact cataacacat gtcattcctc ttcatctgaa attcagtatg gccctaaagt
     2881 gtcataaaga cattcctgaa ggtttttttt attattatta ttatattttt actttcttga
     2941 aaggagagag ggtggggcat gcagtagaat ttgctctctg gcctctccct tttttttaag
     3001 aaaaaaaagg ggggtggcta attcttcctt ctagcaatgg cttcagcagc ccgttctctt
     3061 tccctttaca ggcgagaaac cttaccactg tgactgggac ggctgtgggt ggaaattcgc
     3121 ccgctccgat gaactgacca ggcactaccg caaacacaca gggcaccggc cctttcagtg
     3181 ccagaagtgt gacagggcct tttccaggtc ggaccacctt gccttacaca tgaagaggca
     3241 ctttgaggga agaggtagcc tgctaacttg cggggacgta gaggaaaatc ctggtcccat
     3301 aacttcgtat agcatacatt atacgaagtt atcaatgggc ccaaaaaaga aaagaaaagt
     3361 tgaattcatg gccagaaaag tggacctgac cagttgtgac cgagagccga tacacattcc
     3421 tggctctatc cagccatgtg ggtgtttgct ggcctgcgat gcccaggcag tcaggattac
     3481 tcgaattacc gaaaatgcgg gtgccttctt tggccgtgaa acgccccgtg tcggcgaact
     3541 cttagccgat tatttcggag aaaccgaggc tcacgcactt aggaacgcac tggcgcaatc
     3601 aagcgatccc aaacgccctg ctctcatctt tgggtggaga gatggactga caggccgtac
     3661 tttcgacata agccttcacc ggcatgacgg aaccagtata attgagtttg aaccggccgc
     3721 cgctgagcaa gcagacaacc cccttcgcct gaccagacaa attatcgccc gcactaagga
     3781 gctgaaatcg ctggaagaaa tggccgctcg tgttcccagg tatcttcagg caatgctggg
     3841 ttatcatagg gtaatgctct acagattcgc cgacgatggc tctggtatgg taatagggga
     3901 ggccaagcga tccgacctgg agtcttttct ggggcagcat ttccccgcat ctctagtacc
     3961 ccagcaggca aggcttttgt acctcaagaa tgccatccgc gtggtatcgg actcccgcgg
     4021 tattagtagc aggatcgttc cagaacacga cgcctctgga gctgcgctgg atctgtcttt
     4081 cgctcacctc cgatctatca gcccttgtca tttagagttt ctgcgaaata tgggggtcag
     4141 cgcaagtatg tccctgagca taataatcga tggaacacta tggggcctaa taatttgcca
     4201 tcattacgaa cctagagcag taccaatggc acagcgagtg gccgccgaaa tgttcgccga
     4261 ttttttgtct cttcatttta ctgccgctca ccatcagcgc ggccacggga ctggctcgac
     4321 aggttctggc agtagtggga ctgcttcatc agaagataat aacatggcca tggccagaaa
     4381 agtggacctg accagttgtg accgagagcc gatacacatt cctggctcta tccagccatg
     4441 tgggtgtttg ctggcctgcg atgcccaggc agtcaggatt actcgaatta ccgaaaatgc
     4501 gggtgccttc tttggccgtg aaacgccccg tgtcggcgaa ctcttagccg attatttcgg
     4561 agaaaccgag gctcacgcac ttaggaacgc actggcgcaa tcaagcgatc ccaaacgccc
     4621 tgctctcatc tttgggtgga gagatggact gacaggccgt actttcgaca taagccttca
     4681 ccggcatgac ggaaccagta taattgagtt tgaaccggcc gccgctgagc aagcagacaa
     4741 cccccttcgc ctgaccagac aaattatcgc ccgcactaag gagctgaaat cgctggaaga
     4801 aatggccgct cgtgttccca ggtatcttca ggcaatgctg ggttatcata gggtaatgct
     4861 ctacagattc gccgacgatg gctctggtat ggtaataggg gaggccaagc gatccgacct
     4921 ggagtctttt ctggggcagc atttccccgc atctctagta ccccagcagg caaggctttt
     4981 gtacctcaag aatgccatcc gcgtggtatc ggactcccgc ggtattagta gcaggatcgt
     5041 tccagaacac gacgcctctg gagctgcgct ggatctgtct ttcgctcacc tccgatctat
     5101 cagcccttgt catttagagt ttctgcgaaa tatgggggtc agcgcaagta tgtccctgag
     5161 cataataatc gatggaacac tatggggcct aataatttgc catcattacg aacctagagc
     5221 agtaccaatg gcacagcgag tggccgccga aatgttcgcc gattttttgt ctcttcattt
     5281 tactgccgct caccatcagc gcggatccgc gtttaaactc gagtctcacg gcttccctcc
     5341 cgaggtggag gagcaggccg ccggcaccct gcccatgagc tgcgcccagg agagcggcat
     5401 ggatagacac cctgctgctt gcgccagcgc caggatcaac gtcttgaagt tcctattctc
     5461 tagaaagtat aggaacttct agatcccacg tagtggatgt gacccacact gccaggagag
     5521 agagttcagt attttttttt ctaacctttc acactgtctt cccacgaggg gaggagccca
     5581 gctggcaagc gctacaatca tggtcaagtt cccagcaagt cagcttgtga atggataatc
     5641 aggagaaagg aagagttcaa gagacaaaac agaaatacta aaaacaaaca aacaaaaaaa
     5701 caaacaaaaa aaacaagaaa aaaaaatcac agaacagatg gggtctgata ctggatggat
     5761 cttctatcat tccaatacca aatccaactt gaacatgccc ggacttacaa aatgccaagg
     5821 ggtgactgga agtttgtgga tatcagggta tacactaaat cagtgagctt ggggggaggg
     5881 aagaccagga ttcccttgaa ttgtgtttcg atgatgcaat acacacgtaa agatcacctt
     5941 gtatgctctt tgccttctta aaaaaaaaaa aagccattat tgtgtcggag gaagaggaag
     6001 cgattcaggt acagaacatg ttctaacagc ctaaatgatg gtgcttggtg agtcgtggtt
     6061 ctaaaggtac caaacggggg agccaaagtt ctccaactgc tgcatacttt tgacaaggaa
     6121 aatctagttt tgtcttccga tctacattga tgacctaagc caggtaaata agcctggttt
     6181 atttctgtaa catttttatg cagacagtct gttatgcact gtggtttcag atgtgcaata
     6241 atttgtacaa tggtttattc ccaagtatgc ctttaagcag aacaaatgtg tttttctata
     6301 tagttccttg ccttaataaa tatgtaatat aaatttaagc aaacttctat tttgtatatt
     6361 tgtaaactac aaagtaaaaa aaaatgaaca ttttgtggag tttgtatttt gcatactcaa
     6421 ggtgagaaat aagaagctca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     6481 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     6541 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     6601 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     6661 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     6721 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     6781 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     6841 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     6901 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     6961 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     7021 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     7081 tacaatttag
//
