LOCUS       sequence-233449-        7055 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Gsk3b 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 7055)
  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 7055)
  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-Gsk3b-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7055
                     /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             1627..1680
                     /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(1681..1714)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1723..1743
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3706..3825
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3828..3861
                     /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(4867..4887)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4867..4885)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4906..4922)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4906..4922)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4919..4941)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4930..4946
                     /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(4954..4960,4961..4978,4979..4984))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4999..5020
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5137..5154)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5308..5896)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5388..5407)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6067..6858,6859..6927))
                     /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     6690..6709
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6928..7032)
                     /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 tgggtaccgg
      661 gccccccctc gagtaattaa tgcagcctgt gcctgaattc taaaaatgat gaagaaagcc
      721 ctgatgtgat ctgtcttcta gacaggaatt catgttgagt tagtagtaca ccaaatttag
      781 gtggctgtga acagaaaact cccttctagt agggcatgtc taaggaagtc atggttgaag
      841 ccttagacct gactcttcag tgctgagaaa gccgcctgct gccttcggaa ggaggctctg
      901 gttcatgtgc catgcacctg tgagctagat gtttccgtgc tctggcagcg tgactctgtg
      961 gtatctataa ggaagtagaa ctaaaagcat tcccctgttg actgccccct agtgggctaa
     1021 aggaatggaa gggcagatta tttgaggaac aggaaaatgc agagttcaga gaggaagtgc
     1081 agtttttaga aagtggtcgt gaagttgata aactgaacag cttaccctta agtggtaagg
     1141 ttcctatgtg ttcttggcat ctgctgtgtc ttcacttgtc caggccctta gcagatggcc
     1201 tgttggaaca cggctcagga tctttagcta gtgtcattta ttttcagtaa gctagactcc
     1261 aacctgagta ctgccggtca gtactagcta tagcaaagcc ttgacagaag tgacctcaaa
     1321 ttcctgagtc tgtgatgcat taagtgtact gccacacgca catacaccac acacgcatgt
     1381 gcacgcacac ttgtgccatc actgagctcc tagcagaaag gaagtcctgg tgctgtgtgt
     1441 ttgaacccac ataggcaatg acagctctgg taggcctcag cactgtgcgg tgagtggtgt
     1501 acttgtgctc tttgcagtgt tataacgttg ctcacgcttt cttgggcctt tggatttgca
     1561 gatactaatg ctggagaccg tggacagacc aataacgccg cttctgcatc agcttccaac
     1621 tccaccgagg gaagaggtag cctgctaact tgcggggacg tagaggaaaa tcctggtccc
     1681 ataacttcgt atagcataca ttatacgaag ttatcaatgg gcccaaaaaa gaaaagaaaa
     1741 gttgaattca tggccagaaa agtggacctg accagttgtg accgagagcc gatacacatt
     1801 cctggctcta tccagccatg tgggtgtttg ctggcctgcg atgcccaggc agtcaggatt
     1861 actcgaatta ccgaaaatgc gggtgccttc tttggccgtg aaacgccccg tgtcggcgaa
     1921 ctcttagccg attatttcgg agaaaccgag gctcacgcac ttaggaacgc actggcgcaa
     1981 tcaagcgatc ccaaacgccc tgctctcatc tttgggtgga gagatggact gacaggccgt
     2041 actttcgaca taagccttca ccggcatgac ggaaccagta taattgagtt tgaaccggcc
     2101 gccgctgagc aagcagacaa cccccttcgc ctgaccagac aaattatcgc ccgcactaag
     2161 gagctgaaat cgctggaaga aatggccgct cgtgttccca ggtatcttca ggcaatgctg
     2221 ggttatcata gggtaatgct ctacagattc gccgacgatg gctctggtat ggtaataggg
     2281 gaggccaagc gatccgacct ggagtctttt ctggggcagc atttccccgc atctctagta
     2341 ccccagcagg caaggctttt gtacctcaag aatgccatcc gcgtggtatc ggactcccgc
     2401 ggtattagta gcaggatcgt tccagaacac gacgcctctg gagctgcgct ggatctgtct
     2461 ttcgctcacc tccgatctat cagcccttgt catttagagt ttctgcgaaa tatgggggtc
     2521 agcgcaagta tgtccctgag cataataatc gatggaacac tatggggcct aataatttgc
     2581 catcattacg aacctagagc agtaccaatg gcacagcgag tggccgccga aatgttcgcc
     2641 gattttttgt ctcttcattt tactgccgct caccatcagc gcggccacgg gactggctcg
     2701 acaggttctg gcagtagtgg gactgcttca tcagaagata ataacatggc catggccaga
     2761 aaagtggacc tgaccagttg tgaccgagag ccgatacaca ttcctggctc tatccagcca
     2821 tgtgggtgtt tgctggcctg cgatgcccag gcagtcagga ttactcgaat taccgaaaat
     2881 gcgggtgcct tctttggccg tgaaacgccc cgtgtcggcg aactcttagc cgattatttc
     2941 ggagaaaccg aggctcacgc acttaggaac gcactggcgc aatcaagcga tcccaaacgc
     3001 cctgctctca tctttgggtg gagagatgga ctgacaggcc gtactttcga cataagcctt
     3061 caccggcatg acggaaccag tataattgag tttgaaccgg ccgccgctga gcaagcagac
     3121 aacccccttc gcctgaccag acaaattatc gcccgcacta aggagctgaa atcgctggaa
     3181 gaaatggccg ctcgtgttcc caggtatctt caggcaatgc tgggttatca tagggtaatg
     3241 ctctacagat tcgccgacga tggctctggt atggtaatag gggaggccaa gcgatccgac
     3301 ctggagtctt ttctggggca gcatttcccc gcatctctag taccccagca ggcaaggctt
     3361 ttgtacctca agaatgccat ccgcgtggta tcggactccc gcggtattag tagcaggatc
     3421 gttccagaac acgacgcctc tggagctgcg ctggatctgt ctttcgctca cctccgatct
     3481 atcagccctt gtcatttaga gtttctgcga aatatggggg tcagcgcaag tatgtccctg
     3541 agcataataa tcgatggaac actatggggc ctaataattt gccatcatta cgaacctaga
     3601 gcagtaccaa tggcacagcg agtggccgcc gaaatgttcg ccgatttttt gtctcttcat
     3661 tttactgccg ctcaccatca gcgcggatcc gcgtttaaac tcgagtctca cggcttccct
     3721 cccgaggtgg aggagcaggc cgccggcacc ctgcccatga gctgcgccca ggagagcggc
     3781 atggatagac accctgctgc ttgcgccagc gccaggatca acgtcttgaa gttcctattc
     3841 tctagaaagt ataggaactt ctagtgaaca gcccccagga gccagctgcg cgggaaagac
     3901 cagcacttac ttgagtgcca ctcagcaaca ctggtcacgt ttggaaagaa aattaaaaag
     3961 aggaaaacaa aaacaaaaac aaaaaaaccc tgttcatttt agtgttcaat tttttttatt
     4021 gttgttctta tttaaccttg taaaatatct atataaatac aaaccagttt cattgtattc
     4081 tcacttttca ggagatccaa gaggtgggga gggttgggcg gggaggagca gagcattaaa
     4141 cacaatctct cccacgacaa tcttttcttt tctttttaaa gtcagaagtc cgctattgta
     4201 taccttaaaa accagactcc tgcctcacgc ccccaccgca acagaagaaa acctgttctg
     4261 tgctggttta ttttctggat ttgttttctt ttaaagcctg gcgtaagact ttgatacagt
     4321 gcacagcttg aaattggttg ggagcttagc aggtgtaact caccagggac ttactgtcac
     4381 ttgtacaatg tgtacttatc cttaggattt gaagacttgc ctttggcgtg tgatgtcagg
     4441 tatgacagac aaaaaaaaaa aagacaaaga aaaaccaagg tgtattattg gtagcccagg
     4501 gaggtcacta aaatctgaag ctctaaggga agatcagaga tggattactt aaggtttgtt
     4561 ttgctcttga ccagtgcagg tgttgcagat gccagcttag gaaggctgct gccaaaggag
     4621 agagcgcgac agagtctgtt gggccctgag gtagatcttg gttcacctga ccaaaggccg
     4681 cctgcttctc ccggtattgt tttgagctgg taccctagga tgaggctgga gatggtgtta
     4741 agggggcagc tctgggtgca tcggaaagcg aagaacagga aggtggcagg ccaggggcag
     4801 atctcaccac tgtgagtgag cttgtccaga gcggccgcca ccgcggtgga gctccagctt
     4861 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     4921 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4981 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5041 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5101 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5161 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5221 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5281 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5341 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5401 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5461 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5521 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5581 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5641 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5701 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     5761 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5821 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5881 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5941 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6001 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6061 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6121 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6181 tggccccagt gctgcaatga taccgcggct gccacgctca ccggctccag atttatcagc
     6241 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6301 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6361 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6421 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6481 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6541 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6601 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6661 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6721 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6781 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6841 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6901 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6961 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7021 aggggttccg cgcacatttc cccgaaaagt gccac
//
