LOCUS       sequence-233456-        7121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Kdm1a 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 7121)
  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 7121)
  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-Kdm1a-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7121
                     /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             1657..1710
                     /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(1711..1744)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1753..1773
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3736..3855
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3858..3891
                     /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(4933..4953)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4933..4951)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4972..4988)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4972..4988)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4985..5007)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4996..5012
                     /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(5020..5026,5027..5044,5045..5050))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5065..5086
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5203..5220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5374..5962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5454..5473)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6133..6924,6925..6993))
                     /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     6756..6775
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6994..7098)
                     /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 tgggtacctg
      661 agcctatttg gggaaagacc acaagcttag ggaagaaatg atatctctga caattttaga
      721 cttttattta ttctaccctt aagatacttt tggaataaag tactatttct catacatgag
      781 ctttgtaaat gtagccgaga ttagaaatga atctagagtg tatacaaagt cgctaatttt
      841 tcttgctcac tttttccctt taaaaagtcc tatttgagga ttaggaaaac agctaagcat
      901 ttaagagtgg ctgctagtct tccagaagac ctgagttcag cccccagtac ctatattgga
      961 tggtttacca cgtcttggaa tgatagcttc agggtatctg acattctctt ctatctccct
     1021 aggcatacaa actttttgaa gccttatttt taattgtacc taattttctg gcagcttgta
     1081 aggttgcaaa ggtccaagtt actagggcgt tgtaggtcct gctctttata tagactatgg
     1141 tatatatata ttgttacagt atttctcaat atcataaccc caaggcttgg tatctgctcc
     1201 gaactagaaa gtcaagtaca ttcctatccc taagaaggct ctcccccttg tggttcagag
     1261 gttaggatgc cttgccaatc ttagccagca ggtattacat gaaataaaac tagaatattg
     1321 tgcatcaata aactgatcaa gagacacttt ggtaaactga caaactagtg atggtagcag
     1381 ataggtcttc aaaagtcaac aggaatttag cgatttgaac acaataaagt tatgtttgca
     1441 acttggtgat tcccctacct ctatccccac tagccaatcc caagactctt ctttgctgga
     1501 gaacacacaa tccggaacta cccagctaca gtccatggtg ctctgttgag tgggcttcga
     1561 gaagcaggaa ggattgccga ccagtttttg ggagccatgt acactttgcc tcgtcaggcc
     1621 acaccaggtg tccctgcaca gcagtcccca agtatggagg gaagaggtag cctgctaact
     1681 tgcggggacg tagaggaaaa tcctggtccc ataacttcgt atagcataca ttatacgaag
     1741 ttatcaatgg gcccaaaaaa gaaaagaaaa gttgaattca tggccagaaa agtggacctg
     1801 accagttgtg accgagagcc gatacacatt cctggctcta tccagccatg tgggtgtttg
     1861 ctggcctgcg atgcccaggc agtcaggatt actcgaatta ccgaaaatgc gggtgccttc
     1921 tttggccgtg aaacgccccg tgtcggcgaa ctcttagccg attatttcgg agaaaccgag
     1981 gctcacgcac ttaggaacgc actggcgcaa tcaagcgatc ccaaacgccc tgctctcatc
     2041 tttgggtgga gagatggact gacaggccgt actttcgaca taagccttca ccggcatgac
     2101 ggaaccagta taattgagtt tgaaccggcc gccgctgagc aagcagacaa cccccttcgc
     2161 ctgaccagac aaattatcgc ccgcactaag gagctgaaat cgctggaaga aatggccgct
     2221 cgtgttccca ggtatcttca ggcaatgctg ggttatcata gggtaatgct ctacagattc
     2281 gccgacgatg gctctggtat ggtaataggg gaggccaagc gatccgacct ggagtctttt
     2341 ctggggcagc atttccccgc atctctagta ccccagcagg caaggctttt gtacctcaag
     2401 aatgccatcc gcgtggtatc ggactcccgc ggtattagta gcaggatcgt tccagaacac
     2461 gacgcctctg gagctgcgct ggatctgtct ttcgctcacc tccgatctat cagcccttgt
     2521 catttagagt ttctgcgaaa tatgggggtc agcgcaagta tgtccctgag cataataatc
     2581 gatggaacac tatggggcct aataatttgc catcattacg aacctagagc agtaccaatg
     2641 gcacagcgag tggccgccga aatgttcgcc gattttttgt ctcttcattt tactgccgct
     2701 caccatcagc gcggccacgg gactggctcg acaggttctg gcagtagtgg gactgcttca
     2761 tcagaagata ataacatggc catggccaga aaagtggacc tgaccagttg tgaccgagag
     2821 ccgatacaca ttcctggctc tatccagcca tgtgggtgtt tgctggcctg cgatgcccag
     2881 gcagtcagga ttactcgaat taccgaaaat gcgggtgcct tctttggccg tgaaacgccc
     2941 cgtgtcggcg aactcttagc cgattatttc ggagaaaccg aggctcacgc acttaggaac
     3001 gcactggcgc aatcaagcga tcccaaacgc cctgctctca tctttgggtg gagagatgga
     3061 ctgacaggcc gtactttcga cataagcctt caccggcatg acggaaccag tataattgag
     3121 tttgaaccgg ccgccgctga gcaagcagac aacccccttc gcctgaccag acaaattatc
     3181 gcccgcacta aggagctgaa atcgctggaa gaaatggccg ctcgtgttcc caggtatctt
     3241 caggcaatgc tgggttatca tagggtaatg ctctacagat tcgccgacga tggctctggt
     3301 atggtaatag gggaggccaa gcgatccgac ctggagtctt ttctggggca gcatttcccc
     3361 gcatctctag taccccagca ggcaaggctt ttgtacctca agaatgccat ccgcgtggta
     3421 tcggactccc gcggtattag tagcaggatc gttccagaac acgacgcctc tggagctgcg
     3481 ctggatctgt ctttcgctca cctccgatct atcagccctt gtcatttaga gtttctgcga
     3541 aatatggggg tcagcgcaag tatgtccctg agcataataa tcgatggaac actatggggc
     3601 ctaataattt gccatcatta cgaacctaga gcagtaccaa tggcacagcg agtggccgcc
     3661 gaaatgttcg ccgatttttt gtctcttcat tttactgccg ctcaccatca gcgcggatcc
     3721 gcgtttaaac tcgagtctca cggcttccct cccgaggtgg aggagcaggc cgccggcacc
     3781 ctgcccatga gctgcgccca ggagagcggc atggatagac accctgctgc ttgcgccagc
     3841 gccaggatca acgtcttgaa gttcctattc tctagaaagt ataggaactt ctagtgagac
     3901 agatggttct gaacagagag atccaacggc atgtcatctg ccacgtaagc aagctcttct
     3961 agcaatacta gatcctactg agaaactcca tgtcatcagc tactgggact cctagtttga
     4021 cagcagaggc tggctccttt ggctgacagc aacttaccca ttgatttgga agtacagctc
     4081 cataaagact gctcgagaag caagtggtgt gagataacct cttagtctat ggtgtttgtt
     4141 tgtttttgtt tttttttaat atattttgag aataaaactt taaaataatt ttatatgaaa
     4201 atttattttt aattgtgtgt gtgtgtgtct atgttggaat gcacatttaa agtgggtgcc
     4261 tgtggaggcc aaaggcgctg gtgctggtca caggtggttc taaaccatcc aaaccaccat
     4321 agttgttggg aaccaaactc aggtcctctg caagaagagt gtggttttat ttaaccattg
     4381 aaccatctct ctaacttttg gatcttttgg attaaagttg tatatacaca gtacacaagt
     4441 gctgctccct tggaaatgca aaatcctacc tctggcaata gctcatctgt tctctccctg
     4501 tctgtgagga gggtccgagg gctctcctac acatgcagca ccttttttaa agcttctttt
     4561 ttaaagcttt ttgtgtatca gtgtttgcct atgtgggttt ggtgcccatg agccttcatt
     4621 tgggtgctag gaaccaaacg tgttctctcc aagagctgca actgctctta accatggagc
     4681 tctctctcca gccccactat agttcttaat ggcagtctat taaatgaaag gttacatggg
     4741 aatgtttctt tgcacttctc atgtgctggt gatcatgtgt tggcatagag caccttgtaa
     4801 ttgaactgtc tcctctatag ctgctataat agtacctata ccccaaatat aatctcccca
     4861 tctctaaata tagtatctgt aaagtcttca ctgctagcgg ccgccaccgc ggtggagctc
     4921 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
     4981 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5041 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5101 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5161 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5221 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5281 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5341 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5401 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5461 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5521 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5581 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5641 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5701 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5761 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     5821 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5881 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5941 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6001 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6061 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6121 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6181 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6241 accatctggc cccagtgctg caatgatacc gcggctgcca cgctcaccgg ctccagattt
     6301 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6361 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6421 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6481 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6541 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6601 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6661 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6721 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6781 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6841 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6901 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6961 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7021 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7081 acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
