LOCUS       sequence-252305-        7053 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Npm1 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 7053)
  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 7053)
  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-Npm1-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7053
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             774..827
                     /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(828..861)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             870..890
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2853..2972
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    2975..3008
                     /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(3996..4016)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3996..4014)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4035..4051)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4035..4051)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4048..4070)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4059..4075
                     /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(4083..4089,4090..4107,4108..4113))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4128..4149
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4266..4283)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4437..5025)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4517..4536)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5196..5987,5988..6056))
                     /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     5819..5838
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6057..6147)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6186..6641)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6323..6344)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6535..6554
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6768..6790
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6782..6799
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6783..6799
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6809..6828
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6809..6827
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 catgtcaagt tactgttatg tttattgtgg tattaggata cttgggccat gcaagggcaa
       61 agcactacca gtgatctctt cccagtggta tgaatttaca gtgttaaata gtaaatataa
      121 tgggttaatg ttagaccttc tgaatgaaca gttgatttaa ttggtttcat cagtattggc
      181 tatagaggtg tctattcaaa aagaattaaa aggaacaata tagcagtaga gtattaaatg
      241 atagtattgc ctatttaaat tatagctccc aggtttgtag tcaaaagtgg ttaggattga
      301 ggcagactgt caatgattgg aagtagtgat gtcatcaaat tcagtccttt cgtgttagct
      361 acagattgac ctaagtttta gaattcacca ctgaagagaa tacttgatat ttttaagttt
      421 ttagaaacat actggcttgt cttaagtatg tccctttggg tttttctagg ttgtgatttt
      481 tttttttttg ctttgtaata ctttgcctag actttaaaca ttctcactca aattctgaaa
      541 cctaaatagg gctgacccac aggctgagga ggcaacactg gccataaagt atttaagcca
      601 tttaattaag gaaactcatg ctatgagtta attctgtatt tttcctttgt aaaatgaaaa
      661 atatttaact gaagaaatga aatttgttta attctataaa gttaacattt ctttaccatt
      721 tttttcctcc tttacaggct attcaagatc tctggcagtg gaggaaatct cttgagggaa
      781 gaggtagcct gctaacttgc ggggacgtag aggaaaatcc tggtcccata acttcgtata
      841 gcatacatta tacgaagtta tcaatgggcc caaaaaagaa aagaaaagtt gaattcatgg
      901 ccagaaaagt ggacctgacc agttgtgacc gagagccgat acacattcct ggctctatcc
      961 agccatgtgg gtgtttgctg gcctgcgatg cccaggcagt caggattact cgaattaccg
     1021 aaaatgcggg tgccttcttt ggccgtgaaa cgccccgtgt cggcgaactc ttagccgatt
     1081 atttcggaga aaccgaggct cacgcactta ggaacgcact ggcgcaatca agcgatccca
     1141 aacgccctgc tctcatcttt gggtggagag atggactgac aggccgtact ttcgacataa
     1201 gccttcaccg gcatgacgga accagtataa ttgagtttga accggccgcc gctgagcaag
     1261 cagacaaccc ccttcgcctg accagacaaa ttatcgcccg cactaaggag ctgaaatcgc
     1321 tggaagaaat ggccgctcgt gttcccaggt atcttcaggc aatgctgggt tatcataggg
     1381 taatgctcta cagattcgcc gacgatggct ctggtatggt aataggggag gccaagcgat
     1441 ccgacctgga gtcttttctg gggcagcatt tccccgcatc tctagtaccc cagcaggcaa
     1501 ggcttttgta cctcaagaat gccatccgcg tggtatcgga ctcccgcggt attagtagca
     1561 ggatcgttcc agaacacgac gcctctggag ctgcgctgga tctgtctttc gctcacctcc
     1621 gatctatcag cccttgtcat ttagagtttc tgcgaaatat gggggtcagc gcaagtatgt
     1681 ccctgagcat aataatcgat ggaacactat ggggcctaat aatttgccat cattacgaac
     1741 ctagagcagt accaatggca cagcgagtgg ccgccgaaat gttcgccgat tttttgtctc
     1801 ttcattttac tgccgctcac catcagcgcg gccacgggac tggctcgaca ggttctggca
     1861 gtagtgggac tgcttcatca gaaaataata acatggccat ggccagaaaa gtggacctga
     1921 ccagttgtga ccgagagccg atacacattc ctggctctat ccagccatgt gggtgtttgc
     1981 tggcctgcga tgcccaggca gtcaggatta ctcgaattac cgaaaatgcg ggtgccttct
     2041 ttggccgtga aacgccccgt gtcggcgaac tcttagccga ttatttcgga gaaaccgagg
     2101 ctcacgcact taggaacgca ctggcgcaat caagcgatcc caaacgccct gctctcatct
     2161 ttgggtggag agatggactg acaggccgta ctttcgacat aagccttcac cggcatgacg
     2221 gaaccagtat aattgagttt gaaccggccg ccgctgagca agcagacaac ccccttcgcc
     2281 tgaccagaca aattatcgcc cgcactaagg agctgaaatc gctggaagaa atggccgctc
     2341 gtgttcccag gtatcttcag gcaatgctgg gttatcatag ggtaatgctc tacagattcg
     2401 ccgacgatgg ctctggtatg gtaatagggg aggccaagcg atccgacctg gagtcttttc
     2461 tggggcagca tttccccgca tctctagtac cccagcaggc aaggcttttg tacctcaaga
     2521 atgccatccg cgtggtatcg gactcccgcg gtattagtag caggatcgtt ccagaacacg
     2581 acgcctctgg agctgcgctg gatctgtctt tcgctcacct ccgatctatc agcccttgtc
     2641 atttagagtt tctgcgaaat atgggggtca gcgcaagtat gtccctgagc ataataatcg
     2701 atggaacact atggggccta ataatttgcc atcattacga acctagagca gtaccaatgg
     2761 cacagcgagt ggccgccgaa atgttcgccg attttttgtc tcttcatttt actgccgctc
     2821 accatcagcg cggatccgcg tttaaactcg agtctcacgg cttccctccc gaggtggagg
     2881 agcaggccgc cggcaccctg cccatgagct gcgcccagga gagcggcatg gatagacacc
     2941 ctgctgcttg cgccagcgcc aggatcaacg tcttgaagtt cctattctct agaaagtata
     3001 ggaacttcta gtaagaaaag ggtttaaaca gtttgaaata ttctgtcttc atttctgtaa
     3061 tagttaatat ctggctgtcc tttttataat gcaaagtgag aactttccct actgtgtttg
     3121 ataaatgttg tccaggttca cttgccaaga atgtgttgtc taaaatgcct gtttagtttt
     3181 caaggatgga actccaccct ttacttggtt ttaagtatgt atggaatgtt atgataggac
     3241 atagtaatag tggtcagatg tggaaatggt agggagacaa atatacatgt gaaataaact
     3301 cagtatttta ataaagtagc actgtttcta tttgcttatt taactctcat actttgaagt
     3361 taacgttagt gccctcattt ctgagcagaa tcactgttca tacattccca tttaggaact
     3421 taacacttaa aacacacctt tcctctattg atagccaatc tgttggacat aaccatagga
     3481 tttattcatc ctgttttgct gtggtatatt gtaacaatgc atgggaaaat acatacttaa
     3541 gaaaaaaact cagtggatag tgtctttcac aaacactcag gctatctgta tggatgcttt
     3601 tcttgaatcc ttttaaaatc tggtttggaa ttgagtctaa agttgcctag caggtcatat
     3661 ggggtaggtt aaaagaggta tggtatagtt tgtctattcc tgctcaagtt tgaagtaact
     3721 ttagtcaaag gctgcttcgt gcctgaacct tttttttttt tttttggttt ttcgagacag
     3781 ggcttctctg tatagccctg tcctgtaaac cgggctggcc tcagaaatct gcctgcctct
     3841 gcctcctgag tgctgggatt aaagatgtgt gccaccacgc ctggctcctg aacctcttta
     3901 tagtgaggaa attctataca tataggtgta taaaactatt tggtaacaga ttagactaaa
     3961 atctgcatat taagctggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc
     4021 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4081 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4141 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4201 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4261 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4321 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4381 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4441 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4501 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4561 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4621 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4681 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4741 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4801 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4861 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4921 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4981 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5041 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5101 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5161 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5221 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5281 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcggctg
     5341 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5401 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5461 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5521 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5581 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5641 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5701 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5761 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5821 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5881 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5941 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6001 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6061 ttccttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6121 ttgaatgtat ttagaaaaat aaacaaatag ggattccgcg cacatttccc cgaaaagtgc
     6181 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     6241 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     6301 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     6361 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     6421 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     6481 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     6541 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     6601 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
     6661 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     6721 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     6781 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
     6841 cttaaatagt tggtaaatta tctcttggtt actgaactta gttttactct tagtagtgta
     6901 ttggctaagg aaataggata tggtttttat ttgctccatt tggctgcttt acagtctgat
     6961 gatacttgga ctcttggaag tggtagtatc ttctccagat agcaaaggag ggtaaaattg
     7021 gtagaatcaa cctagatggg ttaaagcctc ttg
//
