LOCUS       sequence-233462-        7136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Ssrp1 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 7136)
  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 7136)
  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-Ssrp1-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7136
                     /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             1677..1730
                     /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(1731..1764)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1773..1793
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3756..3875
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3878..3911
                     /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(4948..4968)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4948..4966)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4987..5003)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4987..5003)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5000..5022)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5011..5027
                     /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(5035..5041,5042..5059,5060..5065))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5080..5101
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5218..5235)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5389..5977)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5469..5488)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6148..6939,6940..7008))
                     /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     6771..6790
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7009..7113)
                     /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 gagctaggag ggagtatgag aaagccatga aagagtatga aggaggaaga
      721 ggggactcat ctaaaaggtg agtgttggga acaaaacggc taataaccgc ttaaagcaga
      781 ttgtgtttgc caggtcagaa tagagaatta ttcggttttg acagcagttt gagtagtgct
      841 tcttaaatcc ctgaatgccg ttgccaagta tgggtgatgc tttcaggttg ggggataaat
      901 ataggccatt ttcctagatc agttttattt gaagtaatat aatagttcag aaagaaaagt
      961 ccacgtgagc ttttaaatac gacaaaagtg tccggtaagt tttgcatgtg agttgcttta
     1021 gactgtgacc ctggtttttc tgggggtact tacttatccg ttggtgcact ttgggctgac
     1081 ggctgacact acatttgtca tggtgagaat agtgtggcca gtactgggaa aagtcctcat
     1141 taaagctagg agatacatcg cctatgtagt aatagagagc ctaatgacct ggtttctttc
     1201 ccatgtgcct aatctaggga caagtctaag aagaaaaaga aagtaaaagc aaagatggaa
     1261 aaaaagtcca ctccttcccg gggctcgtca tccaagtctt catccaggca gttgagtgac
     1321 agcttcaaga gcaaagagtt tgtgtccagt gatgagagct cttcaggcga gaacaagagc
     1381 aaaaagaaga ggaggcggag cgaggtgcgg caggaatgtg gggggttgag ggctgtgtgt
     1441 gtctggggtg ggtcaggtcc cgttggttgg tctgagagga atgagttgga gtgggcgggt
     1501 ggacttgtga ggttttggtg atttcaggag ttgaaggaag agctaggcct tagcagccct
     1561 cttcccacct ggctgggaca catttaccct aaaagaactt tgcttttcag gactctgaag
     1621 aggagctagc cagtacccct ccaagctcag aggactctgc ctcgggatct gatgaagagg
     1681 gaagaggtag cctgctaact tgcggggacg tagaggaaaa tcctggtccc ataacttcgt
     1741 atagcataca ttatacgaag ttatcaatgg gcccaaaaaa gaaaagaaaa gttgaattca
     1801 tggccagaaa agtggacctg accagttgtg accgagagcc gatacacatt cctggctcta
     1861 tccagccatg tgggtgtttg ctggcctgcg atgcccaggc agtcaggatt actcgaatta
     1921 ccgaaaatgc gggtgccttc tttggccgtg aaacgccccg tgtcggcgaa ctcttagccg
     1981 attatttcgg agaaaccgag gctcacgcac ttaggaacgc actggcgcaa tcaagcgatc
     2041 ccaaacgccc tgctctcatc tttgggtgga gagatggact gacaggccgt actttcgaca
     2101 taagccttca ccggcatgac ggaaccagta taattgagtt tgaaccggcc gccgctgagc
     2161 aagcagacaa cccccttcgc ctgaccagac aaattatcgc ccgcactaag gagctgaaat
     2221 cgctggaaga aatggccgct cgtgttccca ggtatcttca ggcaatgctg ggttatcata
     2281 gggtaatgct ctacagattc gccgacgatg gctctggtat ggtaataggg gaggccaagc
     2341 gatccgacct ggagtctttt ctggggcagc atttccccgc atctctagta ccccagcagg
     2401 caaggctttt gtacctcaag aatgccatcc gcgtggtatc ggactcccgc ggtattagta
     2461 gcaggatcgt tccagaacac gacgcctctg gagctgcgct ggatctgtct ttcgctcacc
     2521 tccgatctat cagcccttgt catttagagt ttctgcgaaa tatgggggtc agcgcaagta
     2581 tgtccctgag cataataatc gatggaacac tatggggcct aataatttgc catcattacg
     2641 aacctagagc agtaccaatg gcacagcgag tggccgccga aatgttcgcc gattttttgt
     2701 ctcttcattt tactgccgct caccatcagc gcggccacgg gactggctcg acaggttctg
     2761 gcagtagtgg gactgcttca tcagaagata ataacatggc catggccaga aaagtggacc
     2821 tgaccagttg tgaccgagag ccgatacaca ttcctggctc tatccagcca tgtgggtgtt
     2881 tgctggcctg cgatgcccag gcagtcagga ttactcgaat taccgaaaat gcgggtgcct
     2941 tctttggccg tgaaacgccc cgtgtcggcg aactcttagc cgattatttc ggagaaaccg
     3001 aggctcacgc acttaggaac gcactggcgc aatcaagcga tcccaaacgc cctgctctca
     3061 tctttgggtg gagagatgga ctgacaggcc gtactttcga cataagcctt caccggcatg
     3121 acggaaccag tataattgag tttgaaccgg ccgccgctga gcaagcagac aacccccttc
     3181 gcctgaccag acaaattatc gcccgcacta aggagctgaa atcgctggaa gaaatggccg
     3241 ctcgtgttcc caggtatctt caggcaatgc tgggttatca tagggtaatg ctctacagat
     3301 tcgccgacga tggctctggt atggtaatag gggaggccaa gcgatccgac ctggagtctt
     3361 ttctggggca gcatttcccc gcatctctag taccccagca ggcaaggctt ttgtacctca
     3421 agaatgccat ccgcgtggta tcggactccc gcggtattag tagcaggatc gttccagaac
     3481 acgacgcctc tggagctgcg ctggatctgt ctttcgctca cctccgatct atcagccctt
     3541 gtcatttaga gtttctgcga aatatggggg tcagcgcaag tatgtccctg agcataataa
     3601 tcgatggaac actatggggc ctaataattt gccatcatta cgaacctaga gcagtaccaa
     3661 tggcacagcg agtggccgcc gaaatgttcg ccgatttttt gtctcttcat tttactgccg
     3721 ctcaccatca gcgcggatcc gcgtttaaac tcgagtctca cggcttccct cccgaggtgg
     3781 aggagcaggc cgccggcacc ctgcccatga gctgcgccca ggagagcggc atggatagac
     3841 accctgctgc ttgcgccagc gccaggatca acgtcttgaa gttcctattc tctagaaagt
     3901 ataggaactt ctagtaaagg agggaattcc cacctcgtca cagctccagt ctctcacata
     3961 gtccttggat tctgtgccat ctgagtaact gctcttggtg gcttccactg ccctgaggct
     4021 ttgagggaag cacctaacct ctgctgcttt gcaaggaagg ctccttgcaa tctggagaga
     4081 gactcggtag gagtgtgttg tcttctactc gcagtgcatt gaaggaccca agtcctcagc
     4141 ctactttcct actttctgac tgtagtaaaa gctgcttcct gtcctgttca agttgctgca
     4201 gcaggggtca tgccaagtca ggcctgtagc tcctaatagg ggcctatttc tacttttatt
     4261 ttgtatttct ggtctgtgaa aacaatttaa taaagggaaa tgactttgga aacgatgtcc
     4321 ttatgtctcc tttcccaatt tgcatgtggt gtttaccttc cctgaccggc agggccctcc
     4381 tgcctgccca gctgggaagc aagtctccag gtacatcgtg tcctttccag ccggacaagc
     4441 attggttttc acccttgtct ggggaactgt gccacatgag agtcaagaaa tgagtactga
     4501 tctgattagg aagcctctca tagttcactc cactccatta gccaacagag gggggcggcg
     4561 gcggcggctc tgagctcctt ttaaaatcac cttatgagtc agggtcttac aaggcttgag
     4621 ataatagaat gaggatcgac tttctagcgt agagtggaaa cttgagtcag gaggattgta
     4681 gaaaatgagt tgtgcatgtt tgtgaagtta agtgtaagaa gattctggca agggagggct
     4741 acaccacttt gtgtttggta actgtattta aaagatctta aaggagacat aacaggaggg
     4801 aaaaaactac cccacttgtc acagcggctc tcaatcagaa tttatccacg tgggggtggc
     4861 cacgtcgttt gccgctctca ggaggctcag gtgtaacagg aagactcttc agcggccgcc
     4921 accgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     4981 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5041 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5101 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5161 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5221 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5281 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5341 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5401 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5461 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5521 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5581 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5641 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5701 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5761 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5821 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5881 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5941 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6001 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6061 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6121 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6181 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6241 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcggc tgccacgctc
     6301 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6361 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6421 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6481 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6541 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6601 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6661 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6721 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6781 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6841 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6901 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6961 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7021 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7081 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac
//
