LOCUS       sequence-238895-        7116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Etv5 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 7116)
  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 7116)
  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-Etv5-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7116
                     /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             1678..1731
                     /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(1732..1765)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1774..1794
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3757..3876
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3879..3912
                     /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)"
     regulatory      4161..4170
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4929..4949)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4929..4947)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4968..4984)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4968..4984)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4981..5003)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4992..5008
                     /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(5016..5022,5023..5040,5041..5046))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5061..5082
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5199..5216)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5370..5958)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5450..5469)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6129..6920,6921..6989))
                     /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     6752..6771
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6990..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 tgggtaccgg
      661 gccccccctc gaggtggatt ttaagttcag ggctaacctg agctacatag tgagttccaa
      721 ttcaacagac ccatgcacta aagtgacagg tgtggccaag gtggagtctg gggcctgcaa
      781 cagactgaag ttccacagaa gctggctgat catattctta attggcgagc ttgggagatt
      841 gctcagtagt taaagctcct tccatgcaag agggctggag ttcagatccc cagaacccag
      901 gtaattctag cctgggaagt caggtgattc aatccctaga actatttcag ccaattctag
      961 gcttgacaga agggttcctg cctcaaaggt gaaagaaaca gtgatggaag attcttgact
     1021 tgagtcatgt gccttcgaag aaaacagggc tctgagccct tcgaaatagc acgtccatcc
     1081 tgggaaaacc ataagctctg gaggtcactg gaaatgtggt ctggcatctg cattgtgact
     1141 taagtaccac accagcccct gtcagtacag gtagaatacg gcttccttac tgctcaagga
     1201 gcctgcccct gcatgcattt gaaatccaaa aatttaaatg ctgtctttta aaatactgag
     1261 ctgaggtgtc aacaaaaccc ttggtgggga ataggtccct aagatgcgat tggaataatc
     1321 agctgctact tagccccttt ggcgcttgac tccctgagag gttggcatct tagaagttct
     1381 cattttcttc actggagtcc cctcaccccc gagtgtcgtg ctccagctga gtctcgtgtg
     1441 atctgtcttc tcacgcaggt ggctggggaa cgctacgtct acaaatttgt gtgtgacccg
     1501 gatgcactct tctctatggc cttcccggac aatcagcgcc cgttcctgaa ggcagaatcc
     1561 gaatgccccc tcaatgagga ggacacgctg cctctgactc actttgaaga caaccctgct
     1621 tacctcctgg acatggatcg ctgcagcagc ctcccctaca ccgaaggctt cgcttacgag
     1681 ggaagaggta gcctgctaac ttgcggggac gtagaggaaa atcctggtcc cataacttcg
     1741 tatagcatac attatacgaa gttatcaatg ggcccaaaaa agaaaagaaa agttgaattc
     1801 atggccagaa aagtggacct gaccagttgt gaccgagagc cgatacacat tcctggctct
     1861 atccagccat gtgggtgttt gctggcctgc gatgcccagg cagtcaggat tactcgaatt
     1921 accgaaaatg cgggtgcctt ctttggccgt gaaacgcccc gtgtcggcga actcttagcc
     1981 gattatttcg gagaaaccga ggctcacgca cttaggaacg cactggcgca atcaagcgat
     2041 cccaaacgcc ctgctctcat ctttgggtgg agagatggac tgacaggccg tactttcgac
     2101 ataagccttc accggcatga cggaaccagt ataattgagt ttgaaccggc cgccgctgag
     2161 caagcagaca acccccttcg cctgaccaga caaattatcg cccgcactaa ggagctgaaa
     2221 tcgctggaag aaatggccgc tcgtgttccc aggtatcttc aggcaatgct gggttatcat
     2281 agggtaatgc tctacagatt cgccgacgat ggctctggta tggtaatagg ggaggccaag
     2341 cgatccgacc tggagtcttt tctggggcag catttccccg catctctagt accccagcag
     2401 gcaaggcttt tgtacctcaa gaatgccatc cgcgtggtat cggactcccg cggtattagt
     2461 agcaggatcg ttccagaaca cgacgcctct ggagctgcgc tggatctgtc tttcgctcac
     2521 ctccgatcta tcagcccttg tcatttagag tttctgcgaa atatgggggt cagcgcaagt
     2581 atgtccctga gcataataat cgatggaaca ctatggggcc taataatttg ccatcattac
     2641 gaacctagag cagtaccaat ggcacagcga gtggccgccg aaatgttcgc cgattttttg
     2701 tctcttcatt ttactgccgc tcaccatcag cgcggccacg ggactggctc gacaggttct
     2761 ggcagtagtg ggactgcttc atcagaaaat aataacatgg ccatggccag aaaagtggac
     2821 ctgaccagtt gtgaccgaga gccgatacac attcctggct ctatccagcc atgtgggtgt
     2881 ttgctggcct gcgatgccca ggcagtcagg attactcgaa ttaccgaaaa tgcgggtgcc
     2941 ttctttggcc gtgaaacgcc ccgtgtcggc gaactcttag ccgattattt cggagaaacc
     3001 gaggctcacg cacttaggaa cgcactggcg caatcaagcg atcccaaacg ccctgctctc
     3061 atctttgggt ggagagatgg actgacaggc cgtactttcg acataagcct tcaccggcat
     3121 gacggaacca gtataattga gtttgaaccg gccgccgctg agcaagcaga caaccccctt
     3181 cgcctgacca gacaaattat cgcccgcact aaggagctga aatcgctgga agaaatggcc
     3241 gctcgtgttc ccaggtatct tcaggcaatg ctgggttatc atagggtaat gctctacaga
     3301 ttcgccgacg atggctctgg tatggtaata ggggaggcca agcgatccga cctggagtct
     3361 tttctggggc agcatttccc cgcatctcta gtaccccagc aggcaaggct tttgtacctc
     3421 aagaatgcca tccgcgtggt atcggactcc cgcggtatta gtagcaggat cgttccagaa
     3481 cacgacgcct ctggagctgc gctggatctg tctttcgctc acctccgatc tatcagccct
     3541 tgtcatttag agtttctgcg aaatatgggg gtcagcgcaa gtatgtccct gagcataata
     3601 atcgatggaa cactatgggg cctaataatt tgccatcatt acgaacctag agcagtacca
     3661 atggcacagc gagtggccgc cgaaatgttc gccgattttt tgtctcttca ttttactgcc
     3721 gctcaccatc agcgcggatc cgcgtttaaa ctcgagtctc acggcttccc tcccgaggtg
     3781 gaggagcagg ccgccggcac cctgcccatg agctgcgccc aggagagcgg catggataga
     3841 caccctgctg cttgcgccag cgccaggatc aacgtcttga agttcctatt ctctagaaag
     3901 tataggaact tctagtaagt ttctgaatgg ccgagtagcc acgccctaga gctagcagtc
     3961 ctcatccagg caaccgaggg cagtggtttt gtttgtgttc ttggctgttc ctaaagcttg
     4021 ccctttgagt attatcagga gaacccaagc tgtctctgga ttcgacaccc tttaagacaa
     4081 ataccttggc tcgggaggag tgggaacagg gagagacaaa aaccaccact tcctggggag
     4141 acgtggatac cgtcctcctt gccaccatgg ccagtatgtg cagagcagta ctgcagtgtg
     4201 ttcagggtta gcgcccatga aaggggaccg agtgtatgac aatctgcgtt gatcatggac
     4261 tactaaatgc cttcacacag gaggctttga cttgcacaat tctttgggaa gaaactgcac
     4321 acccatagga cagtcgataa ggctgagtcg gggaggctca gaccattgag gcttgaaatt
     4381 gcaaaactct tgtagctcat tctgaaatct ttcccctgta gtccagaact ggggaggcca
     4441 ggagagccgt tactcactcc atggctctca gaccgcgttc ctctttggcc ttccttccag
     4501 ccgagccgtc accataagta aatcaagtcc cttcctccca gtcacacggc tgagcgcaga
     4561 cggcttgttt taaaaagatg atgaacatga cagtattttg ttttgctttg atttgggggg
     4621 gaagggttca tcttgctttt ggaggttgtt ttggggaaaa aacagtttat ataaactgat
     4681 ttttgtagtt ttggtattta aagcaaaaac aaacaaaaaa caaacccctt tttggtacct
     4741 gcactgcatc cctcgggtgg ggcagtgcct actttggaag atgctgcagc ctgcgagggg
     4801 atctgctggg gcttcccctt cgccacacag aagcctgctt agtgctttct gcaccagaca
     4861 acctgttgag gcgtataacc tgatctgtac atgcggccgc caccgcggtg gagctccagc
     4921 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     4981 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5041 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5101 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5161 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5221 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5281 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5341 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5401 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5461 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5521 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5581 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5641 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5701 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5761 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5821 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5881 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5941 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6001 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6061 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6121 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6181 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6241 tctggcccca gtgctgcaat gataccgcgg ctgccacgct caccggctcc agatttatca
     6301 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6361 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6421 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6481 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6541 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6601 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6661 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6721 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6781 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6841 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6901 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6961 agggcgacac ggaaatgttg aatactcata ctcttccttt tcaatattat tgaagcattt
     7021 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7081 tagggattcc gcgcacattt ccccgaaaag tgccac
//
