LOCUS       sequence-330621-        7407 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nanog targeting vector to insert fluorescent reporter of protein and
            gene expression levels from endogenous locus in mouse embryonic stem
            cells. Please see depositor comments for more detail..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7407)
  AUTHORS   Pokrass MJ, Ryan KA, Xin T, Pielstick B, Timp W, Greco V, Regot S
  TITLE     Cell-Cycle-Dependent ERK Signaling Dynamics Direct Fate 
            Specification in the Mammalian Preimplantation Embryo.
  JOURNAL   Dev Cell. 2020 Nov 9;55(3):328-340.e5. doi: 
            10.1016/j.devcel.2020.09.013. Epub 2020 Oct 21.
  PUBMED    33091369
REFERENCE   2  (bases 1 to 7407)
  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"; doi: 
            "10.1016/j.devcel.2020.09.013"; journalName: "Dev Cell"; date: 
            "2020-11-9- 9"; volume: "55"; issue: "3"; pages: "328-340.e5"
COMMENT     Sequence Label: pMP108 Nanog-Venus-2a-mCherry
FEATURES             Location/Qualifiers
     source          1..7407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..100
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(1098..1100,1101..1103,1104..1814)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1143..1164)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1404..1423)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1751..1772
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1821..1877
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1878..2588
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2023..2041)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2291..2310
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    2621..2654
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2681..2704)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3078..3097
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3159..3962
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3222..3241)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3832..3851
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3994..4011)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4000..4111
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4199..4232
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(5279..5378)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5490..5509)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5501..6310
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVSLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5568..5587)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6400..6988
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6889..6908
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      7153..7239
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      7331..7358
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     7366..7389
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
ORIGIN
        1 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgataagcaa
       61 tgctttttta taatgccaac tttgtacaaa aaagcaggct ccacctatga gacttacgca
      121 acatctgggc tctgcaggtt aagacctggt ttcaaaacca aaggatgaag tgcaagcggt
      181 ggcagaaaaa ccagtggttg aagactagca atggtctgat tcaggtaaca agatgttttc
      241 tctgctttcc tttttccccc tccatcttac catcttattt tatgtgtctg tgccgtgtgt
      301 gtgtgtgtat gtgtgtgtgt ggctgaccta tggctgttgg gtatggatcc cagatttcct
      361 gtaagagcag tcagtggctc tgcctttgct tcccagtgtc atcctgatcc atctatgtcc
      421 attagtcctg tccctttccc atccctcttc ctccacgcac tctttcttgt gatctttggt
      481 tctttgaggc aggctctctc tatgtagccc tggcagtcag agatctgcct gcctagtctc
      541 aggagtgctg gggttaacgg cctgtgcggc catgctaatc ttttcagtat ttctgcagtt
      601 tacatcttta gagtcccctt ttcactcatt gcattaatca cctctttcag aagggctcag
      661 caccagtgga gtatcccagc atccattgca gctatcccca gggctatctg gtgaacgcat
      721 ctggaagcct ttccatgtgg ggcagccaga cttggaccaa cccaacttgg agcagccaga
      781 cctggaccaa cccaacttgg aacaaccaga cctggaccaa cccaacttgg agcagccagg
      841 cctggaccgc tcagtcctgg aacggccagc cttggaatgc tgctccgctc cataacttcg
      901 gggaggactt tctgcagcct tacgtacagt tgcagcaaaa cttctctgcc agtgatttgg
      961 aggtgaattt ggaagccact agggaaagcc atgcgcattt tagcacccca caagccttgg
     1021 aattattcct gaactactct gtgactccac caggtgaaat agggcatggc accggcagca
     1081 ccggcagcgg cagctcaatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1141 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1201 agggcgatgc cacctacggc aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc
     1261 ccgtgccctg gcccaccctc gtgaccaccc tgggctacgg cctgcagtgc ttcgcccgct
     1321 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1381 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1441 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1501 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcaccg
     1561 ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg
     1621 gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1681 tgctgcccga caaccactac ctgagctacc agtccaagct gagcaaagac cccaacgaga
     1741 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1801 acgagctgta caaggctagc gccactaact tctccctgtt gaaacaagca ggggatgtcg
     1861 aagagaatcc cgggccaatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg
     1921 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
     1981 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     2041 agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca
     2101 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
     2161 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     2221 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
     2281 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga
     2341 tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg
     2401 gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc
     2461 ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca
     2521 tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt
     2581 acaagtaatt aattaaccag tgtgctggaa ttcggcttca acaacttcgt ataatgtatg
     2641 ctatacgaag ttatcagtac ttttcccaag gcagtctgga gcatgcgctt tagcagcccc
     2701 gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg
     2761 taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct
     2821 agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt
     2881 agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg
     2941 cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg
     3001 aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag
     3061 gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc
     3121 ctcttcctca tctccgggcc tttcgacctg cagccaatat gggatcggcc attgaacaag
     3181 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3241 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3301 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     3361 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3421 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3481 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3541 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3601 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     3661 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gatgatctcg
     3721 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     3781 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     3841 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     3901 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     3961 gaggggatca attctctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     4021 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     4081 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     4141 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcaac
     4201 aacttcgtat aatgtatgct atacgaagtt atcagtacaa gccgaattct gcagatatcc
     4261 atcggcgcgc cgacttacgc aacatctggg cttaaagtca gggcaaagcc aggttccttc
     4321 cttcttccaa atattttcat atttttttta aagatttatt tattcattat atgtaagtac
     4381 actgtagctg tcttcagaca ctccagaaga gggcgtcaga tcttgttacg tatggttgtg
     4441 agccaccatg tggttgctgg gatttgaact cctgaccttc ggaagagcag tcgggtgctc
     4501 ttatccactg agccatctca ccagcccctg gtttattttt ttaattatta tttgcttttt
     4561 gtttatcaag acagggtttc tctgcatagc tctaattgtc tttgaactag ctctgcagac
     4621 cagcctggcc ttgaactcag agatctgccc acttatcttt gcctcctgaa tgctgggacc
     4681 aaaggtggca taccaccaca cctggcatat atattgttta tttctatttc tatttttatt
     4741 ggtgccagag caaacctagg acttagaaca tgctgggcac caactcaact tctgagctct
     4801 atttacaact tggtgtgtta gtgtatttgt cttagttctg aatttgtcct ttttttagtg
     4861 ttaactctag gctttggaga cagtgaggtg catatactct ctccttccca agaataagtg
     4921 cttgaacacc cttacccacg cccacccacc catgctagtc ttttttctta gaagcgtggg
     4981 tcttggtata cactgtgtca ttttgagggg tgaggtttaa aagtatatac aaagtataac
     5041 gatatggtgg ctactctcga ggatgagaca gaaggaccag gagtttgagg gtagctcaga
     5101 tatgcaataa gttcaaggcc aacctgtact atgtttaaat agtaagacag catctcgata
     5161 aaataataaa actaaagtct caacaaaata aaagctttca cctattaagg tgcttgcttg
     5221 tccttggagt ccccagatgt tgcgtaagtc tcatagcggc cgcactcgag atatctagac
     5281 ccagctttct tgtacaaagt tggcattata agaaagcatt gcttatcaat ttgttgcaac
     5341 gaacaggtca ctatcagtca aaataaaatc attatttgcc atccagctgc agctctggcc
     5401 cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat
     5461 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     5521 aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     5581 tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc
     5641 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     5701 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     5761 tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt
     5821 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgtccctgcg
     5881 ccggttgcat tcgattcctg tctgtaattg tccttttaac agcgatcgcg tatttcgtct
     5941 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     6001 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     6061 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     6121 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     6181 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     6241 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     6301 gtttttctaa tcagaattgg ttaattggtt gtaacattat tcagattggg ccccgttcca
     6361 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6421 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6481 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6541 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6601 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6661 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6721 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6781 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6841 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6901 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6961 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7021 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     7081 taaccgtatt accgctagca tggatctcgg ggacgtctaa ctactaagcg agagtaggga
     7141 actgccaggc atcaaataaa acgaaaggct cagtcggaag actgggcctt tcgttttatc
     7201 tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac
     7261 gttgtgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat
     7321 caaactaagc agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttcct
     7381 gttagttagt tacttaagct cgggccc
//
