LOCUS       sequence-273928-        6710 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor to generate i-WT-r system .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6710)
  AUTHORS   Oksuz O, Narendra V, Lee CH, Descostes N, LeRoy G, Raviram R, 
            Blumenberg L, Karch K, Rocha PP, Garcia BA, Skok JA, Reinberg D
  TITLE     Capturing the Onset of PRC2-Mediated Repressive Domain Formation.
  JOURNAL   Mol Cell. 2018 Jun 21;70(6):1149-1162.e5. doi: 
            10.1016/j.molcel.2018.05.023.
  PUBMED    29932905
REFERENCE   2  (bases 1 to 6710)
  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.molcel.2018.05"; journalName: "Mol Cell"; date: 
            "2018-06-21- 21"; volume: "70"; issue: "6"; pages: "1149-1162.e5"
COMMENT     Sequence Label: A365Y-GFP Donor for iWTr EED
FEATURES             Location/Qualifiers
     source          1..6710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..46
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     38..55
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     39..55
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     62..81
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        62..80
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     154..170
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     154..170
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    complement(999..1032)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1577..1600
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1610..1636
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1652..1705
                     /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"
     CDS             join(1706..1708,1709..2419)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1706..1708,1709..2419)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1748..1769)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2009..2028)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2356..2377
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2447..2464)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2453..2660
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    2661..2694
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3297..3313)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3298..3314)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3458..3474)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3458..3474)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3471..3493)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3482..3498
                     /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(3506..3512,3513..3530,3531..3536))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3551..3572
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3689..3706)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3860..4448)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3940..3959)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4586..4960)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     regulatory      4957..4966
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(5166..5960)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(5166..5960)
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5277..5296)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5887..5906
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6309..6611)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(6411..6430)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
ORIGIN
        1 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
       61 gtaatacgac tcactatagg gcgaattggg ccctctagat gcatgctcga gcggccgcca
      121 gtgtgatgga tatctgcaga attcaggggc cgctctagaa ctagtggatc ccccgggctg
      181 caggaattcg ataacattca ggccccagta aaaatataac tgtgatacct aatcttgact
      241 accaacttac gtcgtgggac ttaagaaagt gaatgggcag cagaattcac tgctctgttt
      301 ctcaataaag atgcatgcac tagctgcctt acactgcttt ctgccacgac ttccatgcca
      361 tgagactgtc catcaaactg gagccaaatt aactcttaaa ttgccttgct gggcattctg
      421 tctcagcacc aagtaactaa tgcagtaaac acaatgaaag cttaactgac cggctggacg
      481 agcaaagagg agaatcccca atcaagacaa cccgatagaa tatcatccaa caaacaagaa
      541 acaaatggag gaggaagaca gccatctgac aagatctctg acaaaaatct tagtgttaga
      601 gtagggacaa aataagacca gcactgtgtg tgcgcaccaa aaatatctaa tatattaata
      661 atgaggatta gtgtctcaaa ataagggaag aaaacagtgt cattagtgtt atagttcaaa
      721 gtatgagaca aaatatttct ctaatatata attccaccac atattccaga tcaatgaact
      781 acaaataagt ttggggttgg gaggttaatg acaaaatatt acacattaga agctatgcag
      841 gccagagaaa gggcaaatac ttttcaagtc tggggagagg ggtggcactg taaagccaga
      901 aatatgtatt taatagatat attctagagt agagagaaaa actaagaaaa aaacaagaga
      961 atttgttact agtagacctc taatgaatag ctgcaggata ccgttcgtat agcatacatt
     1021 atacgaagtt attctgaatt gcttaattct attgctagtc agatttcttg tcctcaggtg
     1081 taagccttga cttgcaaaac agtttatagt cttggaaagt aggctctctg agctattcta
     1141 aaatggtaac agttttaatt ttactttttg aaatgcagtt ctaattatgt gtctcttatt
     1201 tctcttttag tcttgtgaaa atgccattgt atgctggaaa cctggcaaaa tggaggatga
     1261 tatagataaa attaaaccta gtgagtctaa tgtgactatt cttgggcgat ttgattacag
     1321 ccagtgtgac atttggtata tgaggttttc tatggatttc tggcaaaaga tgcttgcatt
     1381 gggcaatcag gttggcaaac tgtatgtttg ggatttagaa gtagaagatc ctcataaagc
     1441 caaatgcaca acactgaccc atcataaatg tggcgcggct attcgacaaa ccagtttcag
     1501 tagggatagc agcatcctca tagctgtctg cgatgatgcc agcatttggc gatgggatcg
     1561 acttcgagga tcaggcgatt acaaggatga cgacgataag ggctccggat acccatacga
     1621 tgtgccagat tacgctgaat tcggcagtgg agagggcaga ggaagtctgc taacatgcgg
     1681 tgacgtcgag gagaatcctg gcccagtgag caagggcgag gagctgttca ccggggtggt
     1741 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1801 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1861 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1921 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1981 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2041 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2101 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     2161 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     2221 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2281 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     2341 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     2401 catggacgag ctgtacaagg aattctaact agagctcgct gatcagcctc gactgtgcct
     2461 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2521 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2581 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag
     2641 aatagcaggc atgctgggga taccgttcgt ataatgtatg ctatacgaag ttatagcaga
     2701 cagtcccatg ttcactttcc aaatttgttt cactgcaaaa attacacaac ccaatatggt
     2761 tctcaaatat aagttaaata cttaagataa ttagatcata agctggggac aaagcaatga
     2821 caagggaagg gtcctatact tcctctgatt accaatatgc caacatatgg tagtctgaaa
     2881 ggttacatat tcgaaatatg acatctaaag caaagcaacc tctaaagctg ctacacaaag
     2941 aaatatgctc cacacgggcc agcaagatgg cacacaggca aaagtcctcc atgcacaagc
     3001 cttgcaagct gagtgcaatg cccagattct atgcaaaaga gaaaagaccc tcgaaggtag
     3061 cctctgactt ccacacagtg cagcagcaca caagcacctg catacacaga cacacataca
     3121 cattactaaa aagtaagcca gataagctgt acataaagag gtgattctca aattaagtgt
     3181 cacatagttt ctgtacttgg gagactgagg agaagtttga agttagcatg ggctatagca
     3241 agtttaaggc tggcttgggt agatggtgag acaccctgtt tcaacatcaa gcttatcgat
     3301 accgtcgacc tcgagggggg gcccggtacc caattcgccc tatagcctga attccagcac
     3361 actggcggcc gttactagtg gatccgagct cggtaccaag cttgatgcat agcttgagta
     3421 ttctaacgcg tcacctaaat agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3481 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3541 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3601 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3661 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3721 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3781 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3841 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3901 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3961 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4021 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4081 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4141 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4201 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4261 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4321 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4381 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4441 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4501 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4561 attaaaaatg aagttttagc acgtgtcagt cctgctcctc ggccacgaag tgcacgcagt
     4621 tgccggccgg gtcgcgcagg gcgaactccc gcccccacgg ctgctcgccg atctcggtca
     4681 tggccggccc ggaggcgtcc cggaagttcg tggacacgac ctccgaccac tcggcgtaca
     4741 gctcgtccag gccgcgcacc cacacccagg ccagggtgtt gtccggcacc acctggtcct
     4801 ggaccgcgct gatgaacagg gtcacgtcgt cccggaccac accggcgaag tcgtcctcca
     4861 cgaagtcccg ggagaacccg agccggtcgg tccagaactc gaccgctccg gcgacgtcgc
     4921 gcgcggtgag caccggaacg gcactggtca acttggccat ggtggccctc ctcacgtgct
     4981 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5041 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgatgcgg
     5101 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta
     5161 ataattcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg
     5221 gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata
     5281 tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg
     5341 atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg
     5401 gtcacgacga gatcctcgcc gtcgggcatg ctcgccttga gcctggcgaa cagttcggct
     5461 ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc
     5521 cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga
     5581 tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca
     5641 aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc
     5701 gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat
     5761 agccgcgctg cctcgtcttg cagttcattc agggcaccgg acaggtcggt cttgacaaaa
     5821 agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc
     5881 tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc
     5941 aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agagcttgat
     6001 cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt gcagggcttc
     6061 ccaaccttac cagagggcgc cccagctggc aattccggtt cgcttgctgt ccataaaacc
     6121 gcccagtcta gctatcgcca tgtaagccca ctgcaagcta cctgctttct ctttgcgctt
     6181 gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca gcaccgtttc
     6241 tgcggactgg ctttctacgt gaaaaggatc taggtgaaga tcctttttga taatctcatg
     6301 cctgacattt atattcccca gaacatcagg ttaatggcgt ttttgatgtc attttcgcgg
     6361 tggctgagat cagccacttc ttccccgata acggagaccg gcacactggc catatcggtg
     6421 gtcatcatgc gccagctttc atccccgata tgcaccaccg ggtaaagttc acgggagact
     6481 ttatctgaca gcagacgtgc actggccagg gggatcacca tccgtcgccc cggcgtgtca
     6541 ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt ataggtgtaa
     6601 accttaaact gccgtacgta taggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     6661 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
//
