LOCUS       sequence-405576-        5812 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a FLAG tagged human histone H3.3 gene with BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5812)
  TITLE     Castro Lab Plasmids 2023
REFERENCE   2  (bases 1 to 5812)
  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-WT-FLAG-BFP
FEATURES             Location/Qualifiers
     source          1..5812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             159..953
                     /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             159..953
                     /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(213..232)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     823..842
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1141..1160)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1185..1232
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1561..2149
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2050..2069
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2324..2627
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2628..2831
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2782..2802
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2897..2962
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    3470..4019
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3614..3631)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3841..3860
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4022..4024,4025..4027,4028..4741)
                     /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(4067..4088)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    4863..4984
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4900..4919)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4954..4973
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4991..5446)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5128..5149)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5340..5359
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5473..5577
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(5579..5812,1..124)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5579..5770
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5602..5622)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(5787..5812,1..110)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
       61 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
      121 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
      181 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
      241 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
      301 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
      361 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
      421 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
      481 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
      541 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
      601 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
      661 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
      721 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
      781 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
      841 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
      901 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
      961 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     1021 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     1081 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     1141 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     1201 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     1261 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     1321 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     1381 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     1441 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     1501 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1561 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     1621 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     1681 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     1741 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1801 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1861 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1921 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     1981 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2041 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2101 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2161 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2221 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
     2281 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     2341 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     2401 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     2461 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     2521 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     2581 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     2641 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     2701 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     2761 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     2821 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact cagatctcga
     2881 gctcaccatg actagtgact acaaggacca cgacggtgac tacaaggacc acgacatcga
     2941 ctacaaggac gacgacgaca agaccggtac tagtgcccga accaagcaga ctgctcgtaa
     3001 gtccaccggt gggaaagccc cccgcaaaca gctggccacg aaagccgcca ggaaaagcgc
     3061 tccctctacc ggcggggtga agaagcctca tcgctacagg cccgggaccg tggcgcttcg
     3121 agagattcgt cgttatcaga agtcgaccga gctgctcatc cggaagctgc ccttccagag
     3181 gttggtgagg gagatcgcgc aggatttcaa aaccgacctg aggtttcaga gcgcagccat
     3241 cggtgcgctg caggaggcta gcgaagcgta cctggtgggt ctgttcgaag ataccaacct
     3301 gtgtgccatc cacgctaaga gagtcaccat catgcccaaa gacatccagt tggctcgccg
     3361 gatacgggga gagagagctt aagaaggatc cctgcagttc ctgcagtcga cggtaccgcg
     3421 ggcccgggat cccaattgct cgagcgggat caattccgcc ccccccctaa cgttactggc
     3481 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg
     3541 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
     3601 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
     3661 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
     3721 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
     3781 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
     3841 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
     3901 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa
     3961 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgataatac
     4021 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     4081 cggcgacgta aacggccaca agttcagcgt gaggggcgag ggcgagggcg atgccaccaa
     4141 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     4201 cctcgtgacc accctgagcc acggcgtgca gtgcttcgcc cgctaccccg accacatgaa
     4261 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     4321 cttcaaggac gacggcacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     4381 ggtgaaccgc atcgagctga agggcgtcga cttcaaggag gacggcaaca tcctggggca
     4441 caagctggag tacaacttca acagccacaa catctatatc atggccgtca agcagaagaa
     4501 cggcatcaag gtgaacttca agatccgcca caacgtggag gacggcagcg tgcagctcgc
     4561 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacagcca
     4621 ctacctgagc acccagtccg tgctgagcaa agaccccaac gagaagcgcg atcacatggt
     4681 cctgctggag ttccgcaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     4741 agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     4801 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     4861 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4921 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4981 cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     5041 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     5101 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     5161 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     5221 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     5281 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     5341 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     5401 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt
     5461 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5521 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct
     5581 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     5641 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     5701 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     5761 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tc
//
