LOCUS       sequence-233460-        7103 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 7103)
  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 7103)
  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..7103
                     /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..1688
                     /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(1689..1722)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1731..1751
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3714..3833
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3836..3869
                     /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(4915..4935)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4915..4933)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4954..4970)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4954..4970)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4967..4989)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4978..4994
                     /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(5002..5008,5009..5026,5027..5032))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5047..5068
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5185..5202)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5356..5944)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5436..5455)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6115..6906,6907..6975))
                     /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     6738..6757
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6976..7080)
                     /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 tgggtacctt
      661 ataagtagaa aagagccagg ctttcaggga atgaagggct gaagcaacga atagactgag
      721 acagttctgc tggaggaact caggccccta aagccgggag tgctattacc agccctaatc
      781 ccaacacttg ggaggctgag gctggcctgg gctacatgga aagaagaagg gaaggggaaa
      841 ccattcaggt gaaaggattg tcccaggagt gttccctttc tagcacagtg cctggatgta
      901 gtgggtctta gatcgcctca aagcagcctt ttcttctctt taaattcagg gcttaatcta
      961 taattatgct ccctgagcgc catgtccaaa tgtccatcac agaacagggg gataactctg
     1021 catctagaac cgcccagtca tttcacctac acacaactat ttcagagcag tacagggtca
     1081 gaaatgtcaa ctgcatcata tactctgtga aatactcctt gtcatgtgca gtctagggct
     1141 ggattttttt ttccctgccc tgcattacta tacatcctaa atacagaaca taaaggaaaa
     1201 ctaaaaatgt taaagtggag gcctgtgagg taaatgtcag tatttggtat ctgacaattt
     1261 ctagcctttt gggattttat gtgtggccac ttaacgttcg cagggctgtt ttgtttctgc
     1321 tgacttgggc atcactgctg aggctttctt gttgctgcat cccattcatt gtcagcatcg
     1381 ggaacaatgc ctgtgctcgc tgggggcttt aatgtacgta ccctttgttt tgttcctgcc
     1441 cttctttgcc agcaacagcc atcattcagg tcgaagatca gaatacaggt gcaattgaga
     1501 acattatagt tgaagtcaaa aaggagccag atgccgagcc tgcggagggg gaagaagagg
     1561 aggctcaggc agccaccaca gacgccccca acggagacct cacgcctgag atgatcctca
     1621 gcatgatgga ccgggaggga agaggtagcc tgctaacttg cggggacgta gaggaaaatc
     1681 ctggtcccat aacttcgtat agcatacatt atacgaagtt atcaatgggc ccaaaaaaga
     1741 aaagaaaagt tgaattcatg gccagaaaag tggacctgac cagttgtgac cgagagccga
     1801 tacacattcc tggctctatc cagccatgtg ggtgtttgct ggcctgcgat gcccaggcag
     1861 tcaggattac tcgaattacc gaaaatgcgg gtgccttctt tggccgtgaa acgccccgtg
     1921 tcggcgaact cttagccgat tatttcggag aaaccgaggc tcacgcactt aggaacgcac
     1981 tggcgcaatc aagcgatccc aaacgccctg ctctcatctt tgggtggaga gatggactga
     2041 caggccgtac tttcgacata agccttcacc ggcatgacgg aaccagtata attgagtttg
     2101 aaccggccgc cgctgagcaa gcagacaacc cccttcgcct gaccagacaa attatcgccc
     2161 gcactaagga gctgaaatcg ctggaagaaa tggccgctcg tgttcccagg tatcttcagg
     2221 caatgctggg ttatcatagg gtaatgctct acagattcgc cgacgatggc tctggtatgg
     2281 taatagggga ggccaagcga tccgacctgg agtcttttct ggggcagcat ttccccgcat
     2341 ctctagtacc ccagcaggca aggcttttgt acctcaagaa tgccatccgc gtggtatcgg
     2401 actcccgcgg tattagtagc aggatcgttc cagaacacga cgcctctgga gctgcgctgg
     2461 atctgtcttt cgctcacctc cgatctatca gcccttgtca tttagagttt ctgcgaaata
     2521 tgggggtcag cgcaagtatg tccctgagca taataatcga tggaacacta tggggcctaa
     2581 taatttgcca tcattacgaa cctagagcag taccaatggc acagcgagtg gccgccgaaa
     2641 tgttcgccga ttttttgtct cttcatttta ctgccgctca ccatcagcgc ggccacggga
     2701 ctggctcgac aggttctggc agtagtggga ctgcttcatc agaagataat aacatggcca
     2761 tggccagaaa agtggacctg accagttgtg accgagagcc gatacacatt cctggctcta
     2821 tccagccatg tgggtgtttg ctggcctgcg atgcccaggc agtcaggatt actcgaatta
     2881 ccgaaaatgc gggtgccttc tttggccgtg aaacgccccg tgtcggcgaa ctcttagccg
     2941 attatttcgg agaaaccgag gctcacgcac ttaggaacgc actggcgcaa tcaagcgatc
     3001 ccaaacgccc tgctctcatc tttgggtgga gagatggact gacaggccgt actttcgaca
     3061 taagccttca ccggcatgac ggaaccagta taattgagtt tgaaccggcc gccgctgagc
     3121 aagcagacaa cccccttcgc ctgaccagac aaattatcgc ccgcactaag gagctgaaat
     3181 cgctggaaga aatggccgct cgtgttccca ggtatcttca ggcaatgctg ggttatcata
     3241 gggtaatgct ctacagattc gccgacgatg gctctggtat ggtaataggg gaggccaagc
     3301 gatccgacct ggagtctttt ctggggcagc atttccccgc atctctagta ccccagcagg
     3361 caaggctttt gtacctcaag aatgccatcc gcgtggtatc ggactcccgc ggtattagta
     3421 gcaggatcgt tccagaacac gacgcctctg gagctgcgct ggatctgtct ttcgctcacc
     3481 tccgatctat cagcccttgt catttagagt ttctgcgaaa tatgggggtc agcgcaagta
     3541 tgtccctgag cataataatc gatggaacac tatggggcct aataatttgc catcattacg
     3601 aacctagagc agtaccaatg gcacagcgag tggccgccga aatgttcgcc gattttttgt
     3661 ctcttcattt tactgccgct caccatcagc gcggatccgc gtttaaactc gagtctcacg
     3721 gcttccctcc cgaggtggag gagcaggccg ccggcaccct gcccatgagc tgcgcccagg
     3781 agagcggcat ggatagacac cctgctgctt gcgccagcgc caggatcaac gtcttgaagt
     3841 tcctattctc tagaaagtat aggaacttct agtgatgctg gggccttgct cggcaccagg
     3901 actattgggc tgtgtttaaa cggcccaaat cttaattttt ctcttttttt tctttggctt
     3961 tgggaacggc ataattttac accattttac caaacatact gagaacgaaa acttcaagga
     4021 tgatgttaga aaaatgtgat ttaactagaa cttgtttgat gttagcaaat catggaatgt
     4081 tctaagtctc tgagggttta ctgtgaagtg ttgaggacag tgttgatgcc taactagttt
     4141 tcttagatgg aaacagagac attgagccct ctctcttgat cgtaaaccac tccagaacgg
     4201 ccacgggttt cccagagttc tatggtcttc ccaagagaat ttttaattgt aaatgcagac
     4261 ttgggaagga cttggacttt taacctggtt ttgcttttgc ttttcctgac tccctttgct
     4321 cagagtcagt tgcacaccag taggatggca tgctacgatc agtttctgtc ctgaaagctt
     4381 tgcctctttc ttggcaaagt ttctggtatg gtcaagcttg taaataacct tttttacatt
     4441 ttaatctttt ccattaatta agaggttgaa aagaagtaca gtgtaagaaa acccagcatt
     4501 ttaattattt gcaaattaag gtgccacaga cacagactta gtggtatgta aatgttgact
     4561 cggatcacaa tcatgtagca gagggcactg ggcctgttgc ctgccagtca tggacaccca
     4621 tgtgaaaaat cctgatttcc agctcatgga gccagcgttt gaaccttgaa caaataaatt
     4681 tcatttatga ttttctcatc aacattttct ttgctctgtt tgtagctgaa tgttacattt
     4741 ttttaaaaat ctctgttcaa gccagaagag ttgattatga gtggccacag caaccctggc
     4801 aagctgggcc aaaaaatttc agtaggtcag taatttaaac ttcacatctg gaaaaaaaat
     4861 tattaataat gtgaaacaaa ccaacttaaa aagtgagagc tccagctttt gttcccttta
     4921 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4981 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5041 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5101 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5161 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5221 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5281 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5341 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5401 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5461 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5521 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5581 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5641 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5701 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5761 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     5821 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5881 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5941 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6001 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6061 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6121 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6181 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6241 tgcaatgata ccgcggctgc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6301 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6361 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6421 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6481 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6541 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6601 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6661 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6721 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6781 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6841 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6901 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6961 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7021 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7081 cacatttccc cgaaaagtgc cac
//
