LOCUS       sequence-233465-        7153 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Targeting vector for mouse Hes1 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 7153)
  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 7153)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-mHes1-tdiRFP670-PEST
FEATURES             Location/Qualifiers
     source          1..7153
                     /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             797..808
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1687..1740
                     /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(1741..1774)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1783..1803
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3766..3885
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     protein_bind    3888..3921
                     /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(4965..4985)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4965..4983)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5004..5020)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5004..5020)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5017..5039)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5028..5044
                     /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(5052..5058,5059..5076,5077..5082))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5097..5118
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5235..5252)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5406..5994)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5486..5505)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6165..6956,6957..7025))
                     /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     6788..6807
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7026..7130)
                     /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 tgggtacctt
      661 atagccacag ctcccaagtt gttactgctc cggaaatgga gggagaggct gtagccgaga
      721 agggtggtgg gcagcttggc tgtggtagaa gccggtgagg gcgaaaggac gtaagactga
      781 ggaagtttgg ggctggatag aaggtagggg tcagtggttt agcaccggtc caacccctgc
      841 cgcaggaggg gaagtggggt ttggaaggcg ccttgggtgg cattgtccaa ggtcaggtcg
      901 cgcgcgggac taggggtggc agatctggag ctgagatcgg tttagatgga gggagtgtcc
      961 tggcttatgt ccccaaccta gggagaagga gctggctgtt cttagcctac ccaaccctcc
     1021 caaccccagg tggttccgga ggcgcgctgc ggggacctcc cagggcggga ggctgtatgg
     1081 ctccggggcc agggcttttc ggtgacccat cttttctcct tctggcctgc agccgcgctc
     1141 agcacagacc cgagcgtgtt ggggaaatac cgcgccggct tcagcgagtg catgaacgag
     1201 gtgacccgct tcctgtccac gtgtgagggc gttaacaccg aggtgcgcac tcggctgctg
     1261 ggccacctgg ccaactgcat gacccagatc aacgccatga cctaccccgg gcaggcgcac
     1321 cccgccttgc aggcgccgcc gccgccgccc ccgtcaggac ctgccggtcc ccagcacgcg
     1381 ccattcgcgc cgccgccgcc gccgcttgtg cccatccccg ggggcgcggc gccccctccc
     1441 ggcagcgcac cctgcaagtt gggcagccag gctggagagg ctgccaaggt ttttggcggc
     1501 ttccaagtgg tgccggctcc tgacggccaa tttgcctttc tcatccccaa cggggccttc
     1561 gctcacagcg gcccggtcat cccggtctac accagcaaca gtgggacctc ggtgggtcct
     1621 aacgcagtgt caccttccag tggctcctcg ctcacttcgg actccatgtg gagaccgtgg
     1681 cggaacgagg gaagaggtag cctgctaact tgcggggacg tagaggaaaa tcctggtccc
     1741 ataacttcgt atagcataca ttatacgaag ttatcaatgg gcccaaaaaa gaaaagaaaa
     1801 gttgaattca tggccagaaa agtggacctg accagttgtg accgagagcc gatacacatt
     1861 cctggctcta tccagccatg tgggtgtttg ctggcctgcg atgcccaggc agtcaggatt
     1921 actcgaatta ccgaaaatgc gggtgccttc tttggccgtg aaacgccccg tgtcggcgaa
     1981 ctcttagccg attatttcgg agaaaccgag gctcacgcac ttaggaacgc actggcgcaa
     2041 tcaagcgatc ccaaacgccc tgctctcatc tttgggtgga gagatggact gacaggccgt
     2101 actttcgaca taagccttca ccggcatgac ggaaccagta taattgagtt tgaaccggcc
     2161 gccgctgagc aagcagacaa cccccttcgc ctgaccagac aaattatcgc ccgcactaag
     2221 gagctgaaat cgctggaaga aatggccgct cgtgttccca ggtatcttca ggcaatgctg
     2281 ggttatcata gggtaatgct ctacagattc gccgacgatg gctctggtat ggtaataggg
     2341 gaggccaagc gatccgacct ggagtctttt ctggggcagc atttccccgc atctctagta
     2401 ccccagcagg caaggctttt gtacctcaag aatgccatcc gcgtggtatc ggactcccgc
     2461 ggtattagta gcaggatcgt tccagaacac gacgcctctg gagctgcgct ggatctgtct
     2521 ttcgctcacc tccgatctat cagcccttgt catttagagt ttctgcgaaa tatgggggtc
     2581 agcgcaagta tgtccctgag cataataatc gatggaacac tatggggcct aataatttgc
     2641 catcattacg aacctagagc agtaccaatg gcacagcgag tggccgccga aatgttcgcc
     2701 gattttttgt ctcttcattt tactgccgct caccatcagc gcggccacgg gactggctcg
     2761 acaggttctg gcagtagtgg gactgcttca tcagaagata ataacatggc catggccaga
     2821 aaagtggacc tgaccagttg tgaccgagag ccgatacaca ttcctggctc tatccagcca
     2881 tgtgggtgtt tgctggcctg cgatgcccag gcagtcagga ttactcgaat taccgaaaat
     2941 gcgggtgcct tctttggccg tgaaacgccc cgtgtcggcg aactcttagc cgattatttc
     3001 ggagaaaccg aggctcacgc acttaggaac gcactggcgc aatcaagcga tcccaaacgc
     3061 cctgctctca tctttgggtg gagagatgga ctgacaggcc gtactttcga cataagcctt
     3121 caccggcatg acggaaccag tataattgag tttgaaccgg ccgccgctga gcaagcagac
     3181 aacccccttc gcctgaccag acaaattatc gcccgcacta aggagctgaa atcgctggaa
     3241 gaaatggccg ctcgtgttcc caggtatctt caggcaatgc tgggttatca tagggtaatg
     3301 ctctacagat tcgccgacga tggctctggt atggtaatag gggaggccaa gcgatccgac
     3361 ctggagtctt ttctggggca gcatttcccc gcatctctag taccccagca ggcaaggctt
     3421 ttgtacctca agaatgccat ccgcgtggta tcggactccc gcggtattag tagcaggatc
     3481 gttccagaac acgacgcctc tggagctgcg ctggatctgt ctttcgctca cctccgatct
     3541 atcagccctt gtcatttaga gtttctgcga aatatggggg tcagcgcaag tatgtccctg
     3601 agcataataa tcgatggaac actatggggc ctaataattt gccatcatta cgaacctaga
     3661 gcagtaccaa tggcacagcg agtggccgcc gaaatgttcg ccgatttttt gtctcttcat
     3721 tttactgccg ctcaccatca gcgcggatcc gcgtttaaac tcgagtctca cggcttccct
     3781 cccgaggtgg aggagcaggc cgccggcacc ctgcccatga gctgcgccca ggagagcggc
     3841 atggatagac accctgctgc ttgcgccagc gccaggatca acgtcttgaa gttcctattc
     3901 tctagaaagt ataggaactt ctagtgagag cctcaggcca ctgctacccg taaagtccct
     3961 agcccacctc tctcttctga cggacactaa aaacgaactt ggattttagg agagactttt
     4021 ataatttggt ggttattttg ttgctttttt taattctaaa aagttacttt ttgtagagag
     4081 ctgtattaag tgactgacca tgcactgcat ttgtatatat tttatatgtt catattggat
     4141 tgcgcctttg tattataaaa gttgagatga catttcgttt tttacacgag atttcttttt
     4201 ttatgtgatg ccaaagatgt ttgaaaatgc tcttaaaata tcttcctttg gggaagttta
     4261 tttgagaaaa tataataaaa gagtgaaggc ttttatgtct tagaactgat tatttcaaaa
     4321 tttgtaaaaa aaaaaaagtg gtggggcttg aaattcatgt agtttggcaa ttcagaaata
     4381 ggatgtattt tttaaagttt taaacaggaa ccatcaaacc tttcagcaac actcaatgat
     4441 ctaagaagac ctaatttatg agaattcatt tgatggggct ttcctgataa acagtcctta
     4501 tttttttacc ttccaaaggg cggctcccaa ctcactccct gtagtagcca ctggcctcct
     4561 aaagtaaaag cgcgctcaag ccacattgca gccctccagc attgggacct tcatttgtct
     4621 gtccttactc aggcgtagct aggcatcccc tggcacccgt atcacaaagt ttgaaatgcc
     4681 tctcttcacc ctcttcaagc aagctgaaaa ggtcaacaga ttctctctgc cagggtacaa
     4741 ctagttagac tcccaagtgg agcccgggcc agatgattaa cagcagacat atgtttctga
     4801 taagtttctg cgcttgactt taatggagaa agcatgtggg ggttgcgctg gcaggcacac
     4861 gggccgtttt tcccctttta atacaaacag caagcgaccc cttcgcatgg tcatgcccca
     4921 tttccaggca agatgtgggc tccagagagc tccagctttt gttcccttta gtgagggtta
     4981 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5041 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5101 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5161 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5221 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5281 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5341 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5401 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5461 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5521 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5581 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5641 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5701 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5761 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5821 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     5881 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5941 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6001 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6061 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6121 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6181 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6241 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6301 ccgcggctgc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6361 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6421 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6481 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6541 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6601 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6661 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6721 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6781 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6841 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6901 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6961 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7021 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7081 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7141 cgaaaagtgc cac
//
