LOCUS       sequence-273930-        6710 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor to generate i-MT-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: Y365A-GFP Donor for iMTr EED
FEATURES             Location/Qualifiers
     source          1..6710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..73
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     65..82
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     66..82
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     89..108
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        89..107
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     181..197
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     181..197
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    complement(1026..1059)
                     /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             1604..1627
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1637..1663
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1679..1732
                     /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(1733..1735,1736..2446)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1733..1735,1736..2446)
                     /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(1775..1796)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2036..2055)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2383..2404
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2474..2491)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2480..2687
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    2688..2721
                     /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(3324..3340)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3325..3341)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3485..3501)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3485..3501)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3498..3520)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3509..3525
                     /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(3533..3539,3540..3557,3558..3563))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3578..3599
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3716..3733)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3887..4475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3967..3986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4613..4987)
                     /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      4984..4993
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(5193..5987)
                     /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(5193..5987)
                     /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(5304..5323)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5914..5933
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6336..6638)
                     /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(6438..6457)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
ORIGIN
        1 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
       61 acgttgtaaa acgacggcca gtgaattgta atacgactca ctatagggcg aattgggccc
      121 tctagatgca tgctcgagcg gccgccagtg tgatggatat ctgcagaatt caggggccgc
      181 tctagaacta gtggatcccc cgggctgcag gaattcgata acattcaggc cccagtaaaa
      241 atataactgt gatacctaat cttgactacc aacttacgtc gtgggactta agaaagtgaa
      301 tgggcagcag aattcactgc tctgtttctc aataaagatg catgcactag ctgccttaca
      361 ctgctttctg ccacgacttc catgccatga gactgtccat caaactggag ccaaattaac
      421 tcttaaattg ccttgctggg cattctgtct cagcaccaag taactaatgc agtaaacaca
      481 atgaaagctt aactgaccgg ctggacgagc aaagaggaga atccccaatc aagacaaccc
      541 gatagaatat catccaacaa acaagaaaca aatggaggag gaagacagcc atctgacaag
      601 atctctgaca aaaatcttag tgttagagta gggacaaaat aagaccagca ctgtgtgtgc
      661 gcaccaaaaa tatctaatat attaataatg aggattagtg tctcaaaata agggaagaaa
      721 acagtgtcat tagtgttata gttcaaagta tgagacaaaa tatttctcta atatataatt
      781 ccaccacata ttccagatca atgaactaca aataagtttg gggttgggag gttaatgaca
      841 aaatattaca cattagaagc tatgcaggcc agagaaaggg caaatacttt tcaagtctgg
      901 ggagaggggt ggcactgtaa agccagaaat atgtatttaa tagatatatt ctagagtaga
      961 gagaaaaact aagaaaaaaa caagagaatt tgttactagt agacctctaa tgaatagctg
     1021 caggataccg ttcgtatagc atacattata cgaagttatt ctgaattgct taattctatt
     1081 gctagtcaga tttcttgtcc tcaggtgtaa gccttgactt gcaaaacagt ttatagtctt
     1141 ggaaagtagg ctctctgagc tattctaaaa tggtaacagt tttaatttta ctttttgaaa
     1201 tgcagttcta attatgtgtc tcttatttct cttttagtct tgtgaaaatg ccattgtatg
     1261 ctggaaacct ggcaaaatgg aggatgatat agataaaatt aaacctagtg agtctaatgt
     1321 gactattctt gggcgatttg attacagcca gtgtgacatt tgggcaatga ggttttctat
     1381 ggatttctgg caaaagatgc ttgcattggg caatcaggtt ggcaaactgt atgtttggga
     1441 tttagaagta gaagatcctc ataaagccaa atgcacaaca ctgacccatc ataaatgtgg
     1501 cgcggctatt cgacaaacca gtttcagtag ggatagcagc atcctcatag ctgtctgcga
     1561 tgatgccagc atttggcgat gggatcgact tcgaggatca ggcgattaca aggatgacga
     1621 cgataagggc tccggatacc catacgatgt gccagattac gctgaattcg gcagtggaga
     1681 gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc cagtgagcaa
     1741 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1801 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1861 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     1921 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     1981 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     2041 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     2101 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     2161 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     2221 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     2281 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     2341 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     2401 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaaggaat tctaactaga
     2461 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2521 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2581 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2641 gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggatac cgttcgtata
     2701 atgtatgcta tacgaagtta tagcagacag tcccatgttc actttccaaa tttgtttcac
     2761 tgcaaaaatt acacaaccca atatggttct caaatataag ttaaatactt aagataatta
     2821 gatcataagc tggggacaaa gcaatgacaa gggaagggtc ctatacttcc tctgattacc
     2881 aatatgccaa catatggtag tctgaaaggt tacatattcg aaatatgaca tctaaagcaa
     2941 agcaacctct aaagctgcta cacaaagaaa tatgctccac acgggccagc aagatggcac
     3001 acaggcaaaa gtcctccatg cacaagcctt gcaagctgag tgcaatgccc agattctatg
     3061 caaaagagaa aagaccctcg aaggtagcct ctgacttcca cacagtgcag cagcacacaa
     3121 gcacctgcat acacagacac acatacacat tactaaaaag taagccagat aagctgtaca
     3181 taaagaggtg attctcaaat taagtgtcac atagtttctg tacttgggag actgaggaga
     3241 agtttgaagt tagcatgggc tatagcaagt ttaaggctgg cttgggtaga tggtgagaca
     3301 ccctgtttca acatcaagct tatcgatacc gtcgacctcg agggggggcc cggtacccaa
     3361 ttcgccctat agcctgaatt ccagcacact ggcggccgtt actagtggat ccgagctcgg
     3421 taccaagctt gatgcatagc ttgagtattc taacgcgtca cctaaatagc ttggcgtaat
     3481 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     3541 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     3601 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     3661 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3721 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3781 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3841 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3901 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3961 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4021 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4081 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4141 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4201 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4261 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4321 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4381 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4441 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4501 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4561 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttagcacg tgtcagtcct
     4621 gctcctcggc cacgaagtgc acgcagttgc cggccgggtc gcgcagggcg aactcccgcc
     4681 cccacggctg ctcgccgatc tcggtcatgg ccggcccgga ggcgtcccgg aagttcgtgg
     4741 acacgacctc cgaccactcg gcgtacagct cgtccaggcc gcgcacccac acccaggcca
     4801 gggtgttgtc cggcaccacc tggtcctgga ccgcgctgat gaacagggtc acgtcgtccc
     4861 ggaccacacc ggcgaagtcg tcctccacga agtcccggga gaacccgagc cggtcggtcc
     4921 agaactcgac cgctccggcg acgtcgcgcg cggtgagcac cggaacggca ctggtcaact
     4981 tggccatggt ggccctcctc acgtgctatt attgaagcat ttatcagggt tattgtctca
     5041 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5101 ttccccgaaa agtgccacct gatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     5161 taccgcatca ggaaattgta agcgttaata attcagaaga actcgtcaag aaggcgatag
     5221 aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc
     5281 cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg
     5341 tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg
     5401 atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgctc
     5461 gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca
     5521 tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct
     5581 tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc
     5641 atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact
     5701 tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa
     5761 ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcttgcag ttcattcagg
     5821 gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac
     5881 acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc
     5941 acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct
     6001 catcctgtct cttgatcaga gcttgatccc ctgcgccatc agatccttgg cggcaagaaa
     6061 gccatccagt ttactttgca gggcttccca accttaccag agggcgcccc agctggcaat
     6121 tccggttcgc ttgctgtcca taaaaccgcc cagtctagct atcgccatgt aagcccactg
     6181 caagctacct gctttctctt tgcgcttgcg ttttcccttg tccagatagc ccagtagctg
     6241 acattcatcc ggggtcagca ccgtttctgc ggactggctt tctacgtgaa aaggatctag
     6301 gtgaagatcc tttttgataa tctcatgcct gacatttata ttccccagaa catcaggtta
     6361 atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc cccgataacg
     6421 gagaccggca cactggccat atcggtggtc atcatgcgcc agctttcatc cccgatatgc
     6481 accaccgggt aaagttcacg ggagacttta tctgacagca gacgtgcact ggccaggggg
     6541 atcaccatcc gtcgccccgg cgtgtcaata atatcactct gtacatccac aaacagacga
     6601 taacggctct ctcttttata ggtgtaaacc ttaaactgcc gtacgtatag gctgcgcaac
     6661 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
//
