LOCUS       sequence-233446-        7159 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Eif4a1 to insert 2A-tdiRFP670-PEST-2A 
            into immediately downstream of start codon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7159)
  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 7159)
  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-Met-Eif4a1-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7159
                     /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             1685..1738
                     /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(1739..1772)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1781..1801
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3764..3883
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3886..3919
                     /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)"
     CDS             3920..3973
                     /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"
     primer_bind     complement(4971..4991)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4971..4989)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5010..5026)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5010..5026)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5023..5045)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5034..5050
                     /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(5058..5064,5065..5082,5083..5088))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5103..5124
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5241..5258)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5412..6000)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5492..5511)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6171..6962,6963..7031))
                     /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     6794..6813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7032..7136)
                     /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 gagggagaat gttgccttgt ggcctaggtg gagcagccat tcctccttcc
      721 atgttcagag ggcaggaaaa cagtgtgggg gtggtggacg cgcaaaggga ttactgcctg
      781 cccgatcttc aaactttttt ttatatatat aaatatatat aaaaatgcca cggtcctgct
      841 ctggtcaata aaggatcctt tgaagatatg caagtggtgg tcttccttaa ggggcctcaa
      901 aatagtggct gtgctttatc tcaggtcttc ttccagctaa cactgttagc gatccccatt
      961 tcagggcttg agagtccagc tttcgataga tgccatcccc ggggagcaga tggcagtaaa
     1021 tatgaattaa gactcgctta ttcatgtaag gaagcccagg agaaactgga aacatactaa
     1081 atgaaccagt gattgagtgg gacaatcctt gacagcttat ccccctatgc cctttaaccc
     1141 aaaagtgggt tccatccttt ttgtctccag tcggggaaac ttactatcca atgagatatc
     1201 gctacaatat tttcctgaac ggaagaataa ataaaacttg tcctgtaaag aaaacccagg
     1261 taaaggaaag tggcagtcca gactgcccgg aagttcctgg aggctaaggc ctcacccccg
     1321 tcgcttgata ggacctgctg agccacatga ctaaggcacg atcgcctccg cacgtgtaaa
     1381 ggtgctgggt tccaagatgg ctgccccgcc gcgaggcccg acttaagtat gtcacttccg
     1441 caccagcgag aaaggcggac ccttcagcca atgaggccat agggcggggc taggccatga
     1501 tgggctttca aactacccaa tagggcgtcc gaactaaagc gcctacaaag taacgtcacg
     1561 tcgagttgca gagcgccggc aggcggggca gaggtggcca agccaatgcg atggctgggg
     1621 cggggtcgga cgctctataa gttgtcgata ggcgggcact ccgccctaga ttctaaggat
     1681 catggaggga agaggtagcc tgctaacttg cggggacgta gaggaaaatc ctggtcccat
     1741 aacttcgtat agcatacatt atacgaagtt atcaatgggc ccaaaaaaga aaagaaaagt
     1801 tgaattcatg gccagaaaag tggacctgac cagttgtgac cgagagccga tacacattcc
     1861 tggctctatc cagccatgtg ggtgtttgct ggcctgcgat gcccaggcag tcaggattac
     1921 tcgaattacc gaaaatgcgg gtgccttctt tggccgtgaa acgccccgtg tcggcgaact
     1981 cttagccgat tatttcggag aaaccgaggc tcacgcactt aggaacgcac tggcgcaatc
     2041 aagcgatccc aaacgccctg ctctcatctt tgggtggaga gatggactga caggccgtac
     2101 tttcgacata agccttcacc ggcatgacgg aaccagtata attgagtttg aaccggccgc
     2161 cgctgagcaa gcagacaacc cccttcgcct gaccagacaa attatcgccc gcactaagga
     2221 gctgaaatcg ctggaagaaa tggccgctcg tgttcccagg tatcttcagg caatgctggg
     2281 ttatcatagg gtaatgctct acagattcgc cgacgatggc tctggtatgg taatagggga
     2341 ggccaagcga tccgacctgg agtcttttct ggggcagcat ttccccgcat ctctagtacc
     2401 ccagcaggca aggcttttgt acctcaagaa tgccatccgc gtggtatcgg actcccgcgg
     2461 tattagtagc aggatcgttc cagaacacga cgcctctgga gctgcgctgg atctgtcttt
     2521 cgctcacctc cgatctatca gcccttgtca tttagagttt ctgcgaaata tgggggtcag
     2581 cgcaagtatg tccctgagca taataatcga tggaacacta tggggcctaa taatttgcca
     2641 tcattacgaa cctagagcag taccaatggc acagcgagtg gccgccgaaa tgttcgccga
     2701 ttttttgtct cttcatttta ctgccgctca ccatcagcgc ggccacggga ctggctcgac
     2761 aggttctggc agtagtggga ctgcttcatc agaagataat aacatggcca tggccagaaa
     2821 agtggacctg accagttgtg accgagagcc gatacacatt cctggctcta tccagccatg
     2881 tgggtgtttg ctggcctgcg atgcccaggc agtcaggatt actcgaatta ccgaaaatgc
     2941 gggtgccttc tttggccgtg aaacgccccg tgtcggcgaa ctcttagccg attatttcgg
     3001 agaaaccgag gctcacgcac ttaggaacgc actggcgcaa tcaagcgatc ccaaacgccc
     3061 tgctctcatc tttgggtgga gagatggact gacaggccgt actttcgaca taagccttca
     3121 ccggcatgac ggaaccagta taattgagtt tgaaccggcc gccgctgagc aagcagacaa
     3181 cccccttcgc ctgaccagac aaattatcgc ccgcactaag gagctgaaat cgctggaaga
     3241 aatggccgct cgtgttccca ggtatcttca ggcaatgctg ggttatcata gggtaatgct
     3301 ctacagattc gccgacgatg gctctggtat ggtaataggg gaggccaagc gatccgacct
     3361 ggagtctttt ctggggcagc atttccccgc atctctagta ccccagcagg caaggctttt
     3421 gtacctcaag aatgccatcc gcgtggtatc ggactcccgc ggtattagta gcaggatcgt
     3481 tccagaacac gacgcctctg gagctgcgct ggatctgtct ttcgctcacc tccgatctat
     3541 cagcccttgt catttagagt ttctgcgaaa tatgggggtc agcgcaagta tgtccctgag
     3601 cataataatc gatggaacac tatggggcct aataatttgc catcattacg aacctagagc
     3661 agtaccaatg gcacagcgag tggccgccga aatgttcgcc gattttttgt ctcttcattt
     3721 tactgccgct caccatcagc gcggatccgc gtttaaactc gagtctcacg gcttccctcc
     3781 cgaggtggag gagcaggccg ccggcaccct gcccatgagc tgcgcccagg agagcggcat
     3841 ggatagacac cctgctgctt gcgccagcgc caggatcaac gtcttgaagt tcctattctc
     3901 tagaaagtat aggaacttcg agggaagagg tagcctgcta acttgcgggg acgtagagga
     3961 aaatcctggt cccgtgtctg cgagtcagga ttctcggtaa gaaaggcttt tgcaaaaggt
     4021 tatggctact taccccgtag ttccctccaa agggcccgcc gtggggaagt tgagaggccc
     4081 atcgcaccga acctaccagg ttgcgggaga accgagccgg gaggaaccgt ccggggttgg
     4141 gggggtgggg gagattccca tggccgccgc tgaccccagt gtggttacgg ggaaggacga
     4201 cctggctagg gcggagggcg cgagcttcgt cacgaggcct cggcctgtgg gaacgccgta
     4261 cgcgaagctc ggatactgaa aggcaagatg aggtcgcgac ataacgtggg aaagaggcgg
     4321 ctgcgcggcc cggagccggc tggtctggct gcctccctgt gtcgggcagg gcgtgcgagc
     4381 gagggttccg gagagttctg gcggtaccac tcgcgacaag caggggtgcc ccggtcttca
     4441 gcgagtccct ccgtcgcgct tggaatgtgg gtcggggaag aagaaaccgg ccgatcaggg
     4501 cgctggcctt cttgacggcc aggaaagagc ctaacccgga ggagcggccg cgtgaggcac
     4561 ggggaaccca cccggcgcct ggcgggcggg tccattttgc cgcacaagcc gggcaattgg
     4621 caaactgcgg atgggcaggt ccactttcct tcgggggtga gcggcctgag gtatgggagg
     4681 gcgacgccat ttctcgacgg gggcgggcgg gttgcggatt gttccatggc gggggagacg
     4741 ccgccgagtg gcggagcgaa tgagcacatg gccgcttgag agattcccct cccccagcct
     4801 gtttcgcttg tctgttgtgc aatctccccc tttgcgagcg cggctggggc tcattgtgcg
     4861 cgaggccgcc accgcccgcg gcctcccaca tccgggtact gtggaggggg gccggccaga
     4921 gggagtgggg gagggcgcgg gcggcatgac gtgagctcca gcttttgttc cctttagtga
     4981 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5041 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5101 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5161 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5221 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5281 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5341 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5401 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5461 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5521 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5581 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5641 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5701 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5761 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5821 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     5881 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5941 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6001 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6061 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6301 atgataccgc ggctgccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7141 tttccccgaa aagtgccac
//
