LOCUS       sequence-405579-        5807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a FLAG tagged human histone mutant H3.3-G34R with BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5807)
  TITLE     Castro Lab Plasmids 2023
REFERENCE   2  (bases 1 to 5807)
  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"
COMMENT     Sequence Label: pIRES-H3.3-G34R-FLAG-BFP
FEATURES             Location/Qualifiers
     source          1..5807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             162..956
                     /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             162..956
                     /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(216..235)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     826..845
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1144..1163)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1188..1235
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1564..2152
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2053..2072
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2327..2630
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2631..2834
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2785..2805
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2900..2965
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    3468..4017
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3612..3629)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3839..3858
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4020..4022,4023..4025,4026..4739)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(4065..4086)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    4861..4982
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4898..4917)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4952..4971
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4989..5444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5126..5147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5338..5357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5471..5575
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(5577..5807,1..127)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5577..5768
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5600..5620)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(5785..5807,1..113)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
       61 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
      121 ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
      181 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
      241 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
      301 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc
      361 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
      421 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
      481 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
      541 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
      601 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
      661 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga
      721 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
      781 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
      841 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
      901 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
      961 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     1021 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     1081 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct
     1141 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
     1201 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     1261 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     1321 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     1381 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact
     1441 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1501 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1561 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1621 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1681 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     1741 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     1801 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     1861 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     1921 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     1981 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2041 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2101 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2161 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     2221 tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcattagt tattaatagt
     2281 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     2341 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     2401 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     2461 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     2521 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     2581 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     2641 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     2701 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     2761 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     2821 atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg actcagatct
     2881 cgagctcacc atgactagtg actacaagga ccacgacggt gactacaagg accacgacat
     2941 cgactacaag gacgacgacg acaagaccgg tactagtgct cgtacaaagc agactgcccg
     3001 caaatcgacc ggtggtaaag cacccaggaa gcaactggct acaaaagccg ctcgcaagag
     3061 tgcgccctct actggaaggg tgaagaaacc tcatcgttac aggcctggta ctgtggcgct
     3121 ccgtgaaatt agacgttatc agaagtccac tgaacttctg attcgcaaac ttcccttcca
     3181 gcgtctggtg cgagaaattg ctcaggactt taaaacagat ctgcgcttcc agagcgcagc
     3241 tatcggtgct ttgcaggagg caagtgaggc ctatctggtt ggcctttttg aagacaccaa
     3301 cctgtgtgct atccatgcca aacgtgtaac aattatgcca aaagacatcc agctagcacg
     3361 ccgcatacgt ggagaacgtg cttaaggatc ccggatccct gcagtcgacg gtaccgcggg
     3421 cccgggatcc caattgctcg agcgggatca attccgcccc ccccctaacg ttactggccg
     3481 aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc
     3541 gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag
     3601 gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt
     3661 tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa
     3721 ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc
     3781 aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg
     3841 gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat
     3901 gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa
     3961 acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gataatacca
     4021 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     4081 gcgacgtaaa cggccacaag ttcagcgtga ggggcgaggg cgagggcgat gccaccaacg
     4141 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     4201 tcgtgaccac cctgagccac ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc
     4261 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     4321 tcaaggacga cggcacctac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     4381 tgaaccgcat cgagctgaag ggcgtcgact tcaaggagga cggcaacatc ctggggcaca
     4441 agctggagta caacttcaac agccacaaca tctatatcat ggccgtcaag cagaagaacg
     4501 gcatcaaggt gaacttcaag atccgccaca acgtggagga cggcagcgtg cagctcgccg
     4561 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacagccact
     4621 acctgagcac ccagtccgtg ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     4681 tgctggagtt ccgcaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag
     4741 cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta
     4801 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     4861 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4921 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4981 taaggcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     5041 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     5101 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     5161 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     5221 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     5281 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     5341 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     5401 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg
     5461 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5521 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga
     5581 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     5641 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     5701 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     5761 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccag
//
