LOCUS       sequence-238873-        7101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Ctcf 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 7101)
  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 7101)
  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-Ctcf-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..457)
                     /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     351..370
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     584..606
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     598..615
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     599..615
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     625..644
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        625..643
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1634..1687
                     /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(1688..1721)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1730..1750
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3713..3832
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3835..3868
                     /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(4914..4934)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4914..4932)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4953..4969)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4953..4969)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4966..4988)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4977..4993
                     /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(5001..5007,5008..5025,5026..5031))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5046..5067
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5184..5201)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5355..5943)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5435..5454)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6114..6905,6906..6974))
                     /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     6737..6756
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6975..7065)
                     /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 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
      301 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
      361 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
      421 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
      481 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
      541 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
      601 aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt gggtacctta
      661 taagtagaaa agagccaggc tttcagggaa tgaagggctg aagcaacgaa tagactgaga
      721 cagttctgct ggaggaactc aggcccctaa agccgggagt gctattacca gccctaatcc
      781 caacacttgg gaggctgagg ctggcctggg ctacatggaa agaagaaggg aaggggaaac
      841 cattcaggtg aaaggattgt cccaggagtg ttccctttct agcacagtgc ctggatgtag
      901 tgggtcttag atcgcctcaa agcagccttt tcttctcttt aaattcaggg cttaatctat
      961 aattatgctc cctgagcgcc atgtccaaat gtccatcaca gaacaggggg ataactctgc
     1021 atctagaacc gcccagtcat ttcacctaca cacaactatt tcagagcagt acagggtcag
     1081 aaatgtcaac tgcatcatat actctgtgaa atactccttg tcatgtgcag tctagggctg
     1141 gatttttttt tccctgccct gcattactat acatcctaaa tacagaacat aaaggaaaac
     1201 taaaaatgtt aaagtggagg cctgtgaggt aaatgtcagt atttggtatc tgacaatttc
     1261 tagccttttg ggattttatg tgtggccact taacgttcgc agggctgttt tgtttctgct
     1321 gacttgggca tcactgctga ggctttcttg ttgctgcatc ccattcattg tcagcatcgg
     1381 gaacaatgcc tgtgctcgct gggggcttta atgtacgtac cctttgtttt gttcctgccc
     1441 ttctttgcca gcaacagcca tcattcaggt cgaagatcag aatacaggtg caattgagaa
     1501 cattatagtt gaagtcaaaa aggagccaga tgccgagcct gcggaggggg aagaagagga
     1561 ggctcaggca gccaccacag acgcccccaa cggagacctc acgcctgaga tgatcctcag
     1621 catgatggac cgggagggaa gaggtagcct gctaacttgc ggggacgtag aggaaaatcc
     1681 tggtcccata acttcgtata gcatacatta tacgaagtta tcaatgggcc caaaaaagaa
     1741 aagaaaagtt gaattcatgg ccagaaaagt ggacctgacc agttgtgacc gagagccgat
     1801 acacattcct ggctctatcc agccatgtgg gtgtttgctg gcctgcgatg cccaggcagt
     1861 caggattact cgaattaccg aaaatgcggg tgccttcttt ggccgtgaaa cgccccgtgt
     1921 cggcgaactc ttagccgatt atttcggaga aaccgaggct cacgcactta ggaacgcact
     1981 ggcgcaatca agcgatccca aacgccctgc tctcatcttt gggtggagag atggactgac
     2041 aggccgtact ttcgacataa gccttcaccg gcatgacgga accagtataa ttgagtttga
     2101 accggccgcc gctgagcaag cagacaaccc ccttcgcctg accagacaaa ttatcgcccg
     2161 cactaaggag ctgaaatcgc tggaagaaat ggccgctcgt gttcccaggt atcttcaggc
     2221 aatgctgggt tatcataggg taatgctcta cagattcgcc gacgatggct ctggtatggt
     2281 aataggggag gccaagcgat ccgacctgga gtcttttctg gggcagcatt tccccgcatc
     2341 tctagtaccc cagcaggcaa ggcttttgta cctcaagaat gccatccgcg tggtatcgga
     2401 ctcccgcggt attagtagca ggatcgttcc agaacacgac gcctctggag ctgcgctgga
     2461 tctgtctttc gctcacctcc gatctatcag cccttgtcat ttagagtttc tgcgaaatat
     2521 gggggtcagc gcaagtatgt ccctgagcat aataatcgat ggaacactat ggggcctaat
     2581 aatttgccat cattacgaac ctagagcagt accaatggca cagcgagtgg ccgccgaaat
     2641 gttcgccgat tttttgtctc ttcattttac tgccgctcac catcagcgcg gccacgggac
     2701 tggctcgaca ggttctggca gtagtgggac tgcttcatca gaaaataata acatggccat
     2761 ggccagaaaa gtggacctga ccagttgtga ccgagagccg atacacattc ctggctctat
     2821 ccagccatgt gggtgtttgc tggcctgcga tgcccaggca gtcaggatta ctcgaattac
     2881 cgaaaatgcg ggtgccttct ttggccgtga aacgccccgt gtcggcgaac tcttagccga
     2941 ttatttcgga gaaaccgagg ctcacgcact taggaacgca ctggcgcaat caagcgatcc
     3001 caaacgccct gctctcatct ttgggtggag agatggactg acaggccgta ctttcgacat
     3061 aagccttcac cggcatgacg gaaccagtat aattgagttt gaaccggccg ccgctgagca
     3121 agcagacaac ccccttcgcc tgaccagaca aattatcgcc cgcactaagg agctgaaatc
     3181 gctggaagaa atggccgctc gtgttcccag gtatcttcag gcaatgctgg gttatcatag
     3241 ggtaatgctc tacagattcg ccgacgatgg ctctggtatg gtaatagggg aggccaagcg
     3301 atccgacctg gagtcttttc tggggcagca tttccccgca tctctagtac cccagcaggc
     3361 aaggcttttg tacctcaaga atgccatccg cgtggtatcg gactcccgcg gtattagtag
     3421 caggatcgtt ccagaacacg acgcctctgg agctgcgctg gatctgtctt tcgctcacct
     3481 ccgatctatc agcccttgtc atttagagtt tctgcgaaat atgggggtca gcgcaagtat
     3541 gtccctgagc ataataatcg atggaacact atggggccta ataatttgcc atcattacga
     3601 acctagagca gtaccaatgg cacagcgagt ggccgccgaa atgttcgccg attttttgtc
     3661 tcttcatttt actgccgctc accatcagcg cggatccgcg tttaaactcg agtctcacgg
     3721 cttccctccc gaggtggagg agcaggccgc cggcaccctg cccatgagct gcgcccagga
     3781 gagcggcatg gatagacacc ctgctgcttg cgccagcgcc aggatcaacg tcttgaagtt
     3841 cctattctct agaaagtata ggaacttcta gtgatgctgg ggccttgctc ggcaccagga
     3901 ctattgggct gtgtttaaac ggcccaaatc ttaatttttc tctttttttt ctttggcttt
     3961 gggaacggca taattttaca ccattttacc aaacatactg agaacgaaaa cttcaaggat
     4021 gatgttagaa aaatgtgatt taactagaac ttgtttgatg ttagcaaatc atggaatgtt
     4081 ctaagtctct gagggtttac tgtgaagtgt tgaggacagt gttgatgcct aactagtttt
     4141 cttagatgga aacagagaca ttgagccctc tctcttgatc gtaaaccact ccagaacggc
     4201 cacgggtttc ccagagttct atggtcttcc caagagaatt tttaattgta aatgcagact
     4261 tgggaaggac ttggactttt aacctggttt tgcttttgct tttcctgact ccctttgctc
     4321 agagtcagtt gcacaccagt aggatggcat gctacgatca gtttctgtcc tgaaagcttt
     4381 gcctctttct tggcaaagtt tctggtatgg tcaagcttgt aaataacctt ttttacattt
     4441 taatcttttc cattaattaa gaggttgaaa agaagtacag tgtaagaaaa cccagcatgt
     4501 taattatttg caaattaagg tgccacagac acagacttag tggtatgtaa atgttgactc
     4561 ggatcacaat catgtagcag agggcactgg gcctgttgcc tgccagtcat ggacacccat
     4621 gtgaaaaatc ctgatttcca gctcatggag ccagcgtttg aaccttgaac aaataaattt
     4681 catttatgat tttctcatca acattttctt tgctctgttt gtagctgaat gttacatttt
     4741 tttaaaaatc tctgttcaag ccagaagagt tgattatgag tggccacagc aaccctggca
     4801 agctgggcca aaaaatttca gtaggtcagt aatttaaact tcacatctgg aaaaaaaatt
     4861 attaataatg tgaaacaaac caacttaaaa agtgagagct ccagcttttg ttccctttag
     4921 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4981 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5041 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5101 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5161 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5221 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5281 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg tgaaaaggcc
     5341 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5401 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5461 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5521 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5581 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5641 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5701 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5761 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5821 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5881 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5941 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6001 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6061 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6121 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6181 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6241 gcaatgatac cgcggctgcc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6301 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6361 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6421 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6481 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6541 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6601 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6661 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6721 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6781 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6841 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6901 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6961 tgttgaatac tcatactctt ccttttcaat attattgaag catttatcag ggttattgtc
     7021 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg attccgcgca
     7081 catttccccg aaaagtgcca c
//
