LOCUS       sequence-233458-        7118 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 7118)
  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 7118)
  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..7118
                     /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(4930..4950)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4930..4948)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4969..4985)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4969..4985)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4982..5004)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4993..5009
                     /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(5017..5023,5024..5041,5042..5047))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5062..5083
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5200..5217)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5371..5959)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5451..5470)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6130..6921,6922..6990))
                     /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     6753..6772
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6991..7095)
                     /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 atcagaagat 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 tactaaaatg ccttcacaca ggaggctttg acttgcacaa ttctttggga agaaactgca
     4321 cacccatagg acagtcgata aggctgagtc ggggaggctc agaccattga ggcttgaaat
     4381 tgcaaaactc ttgtagctca ttctgaaatc tttcccctgt agtccagaac tggggaggcc
     4441 aggagagccg ttactcactc catggctctc agaccgcgtt cctctttggc cttccttcca
     4501 gccgagccgt caccataagt aaatcaagtc ccttcctccc agtcacacgg ctgagcgcag
     4561 acggcttgtt ttaaaaagat gatgaacatg acagtatttt gttttgcttt gatttggggg
     4621 ggaagggttc atcttgcttt tggaggttgt tttggggaaa aaacagttta tataaactga
     4681 tttttgtagt tttggtattt aaagcaaaaa caaacaaaaa acaaacccct ttttggtacc
     4741 tgcactgcat ccctcgggtg gggcagtgcc tactttggaa gatgctgcag cctgcgaggg
     4801 gatctgctgg ggcttcccct tcgccacaca gaagcctgct tagtgctttc tgcaccagac
     4861 aacctgttga ggcgtataac ctgatctgta catgcggccg ccaccgcggt ggagctccag
     4921 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     4981 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5041 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5101 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5161 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5221 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5281 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5341 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5401 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5461 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5521 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5581 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5641 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5701 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5761 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     5821 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5881 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5941 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6001 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6061 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6121 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6181 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6241 atctggcccc agtgctgcaa tgataccgcg gctgccacgc tcaccggctc cagatttatc
     6301 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6361 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6421 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6481 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6541 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6601 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6661 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6721 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6781 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6841 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6901 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6961 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7021 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7081 aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
