LOCUS       sequence-238899-        7099 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector for mouse Ncapd3 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 7099)
  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 7099)
  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-Ncapd3-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7099
                     /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             1657..1710
                     /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(1711..1744)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1753..1773
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3736..3855
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3858..3891
                     /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(4912..4932)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4912..4930)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4951..4967)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4951..4967)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4964..4986)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4975..4991
                     /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(4999..5005,5006..5023,5024..5029))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5044..5065
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5182..5199)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5353..5941)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5433..5452)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6112..6903,6904..6972))
                     /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     6735..6754
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6973..7063)
                     /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 tgggtaccaa
      661 gatggagaca aggaagaagt gacacttgag tgacaccagg gacagggtgg ttactgattc
      721 catgtaaaga tgtagcccaa agccgggcat ggtggcgcac gtctttaatc ccagcacctg
      781 ggaggcaggg gcaggcggat ttctgagttc aaggccagca tggtctacaa agtgagttca
      841 ggacagccag ggctacacag agaaaccctg tcttgaaaaa ccaaaaaaaa aaaaaaaaaa
      901 agatgtagcc taaggttctg gccagttaaa gccttagaga atcatgtatc aacccttcca
      961 acataaatgg agagggaata aacacaatgg ggtggaagta tggagatcca gtaggtgaga
     1021 agtaatagga aatggaggtc acctgcacag aggcagtcac aggaggagga ggtgagctgg
     1081 gagaagacac tgccatgtca agacaggctg cagtgcaagc gtctgcctaa ccctgagaca
     1141 aacccaagct tccgatgagt ctttgtgagt cacctagggg ctagcagtga agaccagaga
     1201 gagcccatgg caaattccca caagttttat tcccattgtt ctccctttct ccagattcca
     1261 tcagtgacgt cacatttgca gcaggggtca gttacatagg gacaccagca acttttttta
     1321 ccaaaggtaa gttctgggtt ttcttctggt cagcacaact actgcctctg aaattctgag
     1381 aaactaaatt tcttcatgtt tgactccttg tcagagaaac acgaagccca agaacaagga
     1441 agtgacattt tatgtttatc gctgcttgat aaaaggtaag ttttgctagt gggcttcctg
     1501 gttgagctgt tttctggttg agctgttgac tgcatggttt ccttccccag acctccacag
     1561 tctccacagt ggaacgtaaa gtccccagcc aggagtcacg gcagcacaag atccagccgg
     1621 aggtcccttc gtaaggcccc cctgaagaca gccaacgagg gaagaggtag cctgctaact
     1681 tgcggggacg tagaggaaaa tcctggtccc ataacttcgt atagcataca ttatacgaag
     1741 ttatcaatgg gcccaaaaaa gaaaagaaaa gttgaattca tggccagaaa agtggacctg
     1801 accagttgtg accgagagcc gatacacatt cctggctcta tccagccatg tgggtgtttg
     1861 ctggcctgcg atgcccaggc agtcaggatt actcgaatta ccgaaaatgc gggtgccttc
     1921 tttggccgtg aaacgccccg tgtcggcgaa ctcttagccg attatttcgg agaaaccgag
     1981 gctcacgcac ttaggaacgc actggcgcaa tcaagcgatc ccaaacgccc tgctctcatc
     2041 tttgggtgga gagatggact gacaggccgt actttcgaca taagccttca ccggcatgac
     2101 ggaaccagta taattgagtt tgaaccggcc gccgctgagc aagcagacaa cccccttcgc
     2161 ctgaccagac aaattatcgc ccgcactaag gagctgaaat cgctggaaga aatggccgct
     2221 cgtgttccca ggtatcttca ggcaatgctg ggttatcata gggtaatgct ctacagattc
     2281 gccgacgatg gctctggtat ggtaataggg gaggccaagc gatccgacct ggagtctttt
     2341 ctggggcagc atttccccgc atctctagta ccccagcagg caaggctttt gtacctcaag
     2401 aatgccatcc gcgtggtatc ggactcccgc ggtattagta gcaggatcgt tccagaacac
     2461 gacgcctctg gagctgcgct ggatctgtct ttcgctcacc tccgatctat cagcccttgt
     2521 catttagagt ttctgcgaaa tatgggggtc agcgcaagta tgtccctgag cataataatc
     2581 gatggaacac tatggggcct aataatttgc catcattacg aacctagagc agtaccaatg
     2641 gcacagcgag tggccgccga aatgttcgcc gattttttgt ctcttcattt tactgccgct
     2701 caccatcagc gcggccacgg gactggctcg acaggttctg gcagtagtgg gactgcttca
     2761 tcagaaaata ataacatggc catggccaga aaagtggacc tgaccagttg tgaccgagag
     2821 ccgatacaca ttcctggctc tatccagcca tgtgggtgtt tgctggcctg cgatgcccag
     2881 gcagtcagga ttactcgaat taccgaaaat gcgggtgcct tctttggccg tgaaacgccc
     2941 cgtgtcggcg aactcttagc cgattatttc ggagaaaccg aggctcacgc acttaggaac
     3001 gcactggcgc aatcaagcga tcccaaacgc cctgctctca tctttgggtg gagagatgga
     3061 ctgacaggcc gtactttcga cataagcctt caccggcatg acggaaccag tataattgag
     3121 tttgaaccgg ccgccgctga gcaagcagac aacccccttc gcctgaccag acaaattatc
     3181 gcccgcacta aggagctgaa atcgctggaa gaaatggccg ctcgtgttcc caggtatctt
     3241 caggcaatgc tgggttatca tagggtaatg ctctacagat tcgccgacga tggctctggt
     3301 atggtaatag gggaggccaa gcgatccgac ctggagtctt ttctggggca gcatttcccc
     3361 gcatctctag taccccagca ggcaaggctt ttgtacctca agaatgccat ccgcgtggta
     3421 tcggactccc gcggtattag tagcaggatc gttccagaac acgacgcctc tggagctgcg
     3481 ctggatctgt ctttcgctca cctccgatct atcagccctt gtcatttaga gtttctgcga
     3541 aatatggggg tcagcgcaag tatgtccctg agcataataa tcgatggaac actatggggc
     3601 ctaataattt gccatcatta cgaacctaga gcagtaccaa tggcacagcg agtggccgcc
     3661 gaaatgttcg ccgatttttt gtctcttcat tttactgccg ctcaccatca gcgcggatcc
     3721 gcgtttaaac tcgagtctca cggcttccct cccgaggtgg aggagcaggc cgccggcacc
     3781 ctgcccatga gctgcgccca ggagagcggc atggatagac accctgctgc ttgcgccagc
     3841 gccaggatca acgtcttgaa gttcctattc tctagaaagt ataggaactt ctagtgagac
     3901 ctgtggaaat atgtgcatct cctagatgga aggggaaagt gtggtgtggc ctgcacccac
     3961 gtgggtgtgg gagacacgta gctgtgcact gccagctcct gcctaggaca tcagtgtgtc
     4021 tttgtgcagc ttgtgtttgt ctaaccaacc ttcctatttt tatttctctt tctatcatta
     4081 tagctgagat ttgtaaatag agatatgatg tatttagact ttctcacaat aacattaagc
     4141 tatttatact gtgatactct atttgagtat caaacgttct gtggtaagtt acaatatggt
     4201 cttttaagga acacaatttt cgaaaccagc tttcttctgt ataactcaag tgtctatagc
     4261 ttgtgtgact tcagtataat agatatgtcc ccttagctaa tggcaaaaat ttgaaatatt
     4321 tttctattat ttataattac aaatttttat taaaagaggt gtacagtctt tctaaaacaa
     4381 agtaataaat gtcaaatata aatataacct agcatttatt ataatttttc tggctttgga
     4441 gcaaggcttt attgatggtc tgcttaacac tgcattaagt atttaagggt ttaattcatc
     4501 tacccacctg cccttgttcc catagagaac ctttgtctct cttgtgtccc agtagccatc
     4561 cccattagta catcaggtag gtaaagcctg ctcatgatgg catggctgcc ttcttgaccc
     4621 ttagacagag cggtggtgca gatgggtgag actgctgagg agctggaggg ttggggttga
     4681 atgctctgcc tttttggaga caagcatctc atgtattgct cagccttccg agtaacaggg
     4741 acaacaggca tactccacca taccctcctc ctatcccatg ctaacccagg accgtgcacc
     4801 taggcaagcc ctctactact ggtccctgta ctcttgtcat aaagaagctc tgacagttgg
     4861 tggctcgagt gtcacagctg ctcttaagtg aacgagctcc agcttttgtt ccctttagtg
     4921 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4981 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5041 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5101 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5161 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5221 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5281 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5341 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5401 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5461 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5521 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5581 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5641 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5701 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5761 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5821 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5881 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5941 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6001 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6061 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6121 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6181 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6241 aatgataccg cggctgccac gctcaccggc tccagattta tcagcaataa accagccagc
     6301 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6361 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6421 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6481 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6541 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6601 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6661 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6721 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6781 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6841 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6901 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6961 ttgaatactc atactcttcc ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7021 atgagcggat acatatttga atgtatttag aaaaataaac aaatagggat tccgcgcaca
     7081 tttccccgaa aagtgccac
//
