LOCUS       sequence-233455-        7103 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 7103)
  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 7103)
  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..7103
                     /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             1660..1713
                     /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(1714..1747)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1756..1776
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3739..3858
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3861..3894
                     /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(4915..4935)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4915..4933)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4954..4970)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4954..4970)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4967..4989)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4978..4994
                     /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(5002..5008,5009..5026,5027..5032))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5047..5068
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5185..5202)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5356..5944)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5436..5455)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6115..6906,6907..6975))
                     /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     6738..6757
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6976..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 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 aaaagatgta gcctaaggtt ctggccagtt aaagccttag agaatcatgt atcaaccctt
      961 ccaacataaa tggagaggga ataaacacaa tggggtggaa gtatggagat ccagtaggtg
     1021 agaagtaata ggaaatggag gtcacctgca cagaggcagt cacaggagga ggaggtgagc
     1081 tgggagaaga cactgccatg tcaagacagg ctgcagtgca agcgtctgcc taaccctgag
     1141 acaaacccaa gcttccgatg agtctttgtg agtcacctag gggctagcag tgaagaccag
     1201 agagagccca tggcaaattc ccacaagttt tattcccatt gttctccctt tctccagatt
     1261 ccatcagtga cgtcacattt gcagcagggg tcagttacat agggacacca gcaacttttt
     1321 ttaccaaagg taagttctgg gttttcttct ggtcagcaca actactgcct ctgaaattct
     1381 gagaaactaa atttcttcat gtttgactcc ttgtcagaga aacacgaagc ccaagaacaa
     1441 ggaagtgaca ttttatgttt atcgctgctt gataaaaggt aagttttgct agtgggcttc
     1501 ctggttgagc tgttttctgg ttgagctgtt gactgcatgg tttccttccc cagacctcca
     1561 cagtctccac agtggaacgt aaagtcccca gccaggagtc acggcagcac aagatccagc
     1621 cggaggtccc ttcgtaaggc ccccctgaag acagccaacg agggaagagg tagcctgcta
     1681 acttgcgggg acgtagagga aaatcctggt cccataactt cgtatagcat acattatacg
     1741 aagttatcaa tgggcccaaa aaagaaaaga aaagttgaat tcatggccag aaaagtggac
     1801 ctgaccagtt gtgaccgaga gccgatacac attcctggct ctatccagcc atgtgggtgt
     1861 ttgctggcct gcgatgccca ggcagtcagg attactcgaa ttaccgaaaa tgcgggtgcc
     1921 ttctttggcc gtgaaacgcc ccgtgtcggc gaactcttag ccgattattt cggagaaacc
     1981 gaggctcacg cacttaggaa cgcactggcg caatcaagcg atcccaaacg ccctgctctc
     2041 atctttgggt ggagagatgg actgacaggc cgtactttcg acataagcct tcaccggcat
     2101 gacggaacca gtataattga gtttgaaccg gccgccgctg agcaagcaga caaccccctt
     2161 cgcctgacca gacaaattat cgcccgcact aaggagctga aatcgctgga agaaatggcc
     2221 gctcgtgttc ccaggtatct tcaggcaatg ctgggttatc atagggtaat gctctacaga
     2281 ttcgccgacg atggctctgg tatggtaata ggggaggcca agcgatccga cctggagtct
     2341 tttctggggc agcatttccc cgcatctcta gtaccccagc aggcaaggct tttgtacctc
     2401 aagaatgcca tccgcgtggt atcggactcc cgcggtatta gtagcaggat cgttccagaa
     2461 cacgacgcct ctggagctgc gctggatctg tctttcgctc acctccgatc tatcagccct
     2521 tgtcatttag agtttctgcg aaatatgggg gtcagcgcaa gtatgtccct gagcataata
     2581 atcgatggaa cactatgggg cctaataatt tgccatcatt acgaacctag agcagtacca
     2641 atggcacagc gagtggccgc cgaaatgttc gccgattttt tgtctcttca ttttactgcc
     2701 gctcaccatc agcgcggcca cgggactggc tcgacaggtt ctggcagtag tgggactgct
     2761 tcatcagaag ataataacat ggccatggcc agaaaagtgg acctgaccag ttgtgaccga
     2821 gagccgatac acattcctgg ctctatccag ccatgtgggt gtttgctggc ctgcgatgcc
     2881 caggcagtca ggattactcg aattaccgaa aatgcgggtg ccttctttgg ccgtgaaacg
     2941 ccccgtgtcg gcgaactctt agccgattat ttcggagaaa ccgaggctca cgcacttagg
     3001 aacgcactgg cgcaatcaag cgatcccaaa cgccctgctc tcatctttgg gtggagagat
     3061 ggactgacag gccgtacttt cgacataagc cttcaccggc atgacggaac cagtataatt
     3121 gagtttgaac cggccgccgc tgagcaagca gacaaccccc ttcgcctgac cagacaaatt
     3181 atcgcccgca ctaaggagct gaaatcgctg gaagaaatgg ccgctcgtgt tcccaggtat
     3241 cttcaggcaa tgctgggtta tcatagggta atgctctaca gattcgccga cgatggctct
     3301 ggtatggtaa taggggaggc caagcgatcc gacctggagt cttttctggg gcagcatttc
     3361 cccgcatctc tagtacccca gcaggcaagg cttttgtacc tcaagaatgc catccgcgtg
     3421 gtatcggact cccgcggtat tagtagcagg atcgttccag aacacgacgc ctctggagct
     3481 gcgctggatc tgtctttcgc tcacctccga tctatcagcc cttgtcattt agagtttctg
     3541 cgaaatatgg gggtcagcgc aagtatgtcc ctgagcataa taatcgatgg aacactatgg
     3601 ggcctaataa tttgccatca ttacgaacct agagcagtac caatggcaca gcgagtggcc
     3661 gccgaaatgt tcgccgattt tttgtctctt cattttactg ccgctcacca tcagcgcgga
     3721 tccgcgttta aactcgagtc tcacggcttc cctcccgagg tggaggagca ggccgccggc
     3781 accctgccca tgagctgcgc ccaggagagc ggcatggata gacaccctgc tgcttgcgcc
     3841 agcgccagga tcaacgtctt gaagttccta ttctctagaa agtataggaa cttctagtga
     3901 gacctgtgga aatatgtgca tctcctagat ggaaggggaa agtgtggtgt ggcctgcacc
     3961 cacgtgggtg tgggagacac gtagctgtgc actgccagct cctgcctagg acatcagtgt
     4021 gtctttgtgc agcttgtgtt tgtctaacca accttcctat ttttatttct ctttctatca
     4081 ttatagctga gatttgtaaa tagagatatg atgtatttag actttctcac aataacatta
     4141 agctatttat actgtgatac tctatttgag tatcaaacgt tctgtggtaa gttacaatat
     4201 ggtcttttaa ggaacacaat tttcgaaacc agctttcttc tgtataactc aagtgtctat
     4261 agcttgtgtg acttcagtat aatagatatg tccccttagc taatggcaaa aatttgaaat
     4321 atttttctat tatttataat tacaaatttt tattaaaaga ggtgtacagt ctttctaaaa
     4381 caaagtaata aatgtcaaat ataaatataa cctagcattt attataattt ttctggcttt
     4441 ggagcaaggc tttattgatg gtctgcttaa cactgcatta agtatttaag ggtttaattc
     4501 atctacccac ctgcccttgt tcccatagag aacctttgtc tctcttgtgt cccagtagcc
     4561 atccccatta gtacatcagg taggtaaagc ctgctcatga tggcatggct gccttcttga
     4621 cccttagaca gagcggtggt gcagatgggt gagactgctg aggagctgga gggttggggt
     4681 tgaatgctct gcctttttgg agacaagcat ctcatgtatt gctcagcctt ccgagtaaca
     4741 gggacaacag gcatactcca ccataccctc ctcctatccc atgctaaccc aggaccgtgc
     4801 acctaggcaa gccctctact actggtccct gtactcttgt cataaagaag ctctgacagt
     4861 tggtggctcg agtgtcacag ctgctcttaa gtgaacgagc tccagctttt gttcccttta
     4921 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4981 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5041 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5101 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5161 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5221 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5281 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5341 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5401 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5461 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5521 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5581 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5641 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5701 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5761 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     5821 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5881 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5941 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6001 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6061 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6121 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6181 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6241 tgcaatgata ccgcggctgc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6301 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6361 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6421 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6481 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6541 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6601 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6661 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6721 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6781 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6841 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6901 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6961 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7021 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7081 cacatttccc cgaaaagtgc cac
//
