LOCUS       sequence-335371-        7613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for mammalian expression of the Usp9x catalytic domain tagged
            with mCherry, in pEF1a-IRES-Neo backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7613)
  AUTHORS   Macrae TA, Ramalho-Santos M
  TITLE     The deubiquitinase Usp9x regulates PRC2-mediated chromatin 
            reprogramming during mouse development.
  JOURNAL   Nat Commun. 2021 Mar 25;12(1):1865. doi: 10.1038/s41467-021-21910-0.
  PUBMED    33767158
REFERENCE   2  (bases 1 to 7613)
  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"; journalName: "Nat 
            Commun."; date: "2021-03-25"; volume: "12(1):1865. doi"; pages: " 
            10.1038/s41467-021-21910-0"
COMMENT     Sequence Label: pEF1a-Usp9x_CD-mCherry
FEATURES             Location/Qualifiers
     source          1..7613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1163
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1154
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1111..1131
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     intron          1202..1334
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1317..1336
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1379..1397
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2639..3346
                     /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(2784..2802)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3052..3071
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3347..3412
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    3436..4004
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3454..4006
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3598..3615)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3825..3844
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4006..4803
                     /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="MGIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
                     SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
                     QGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
                     ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4063..4082)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4673..4692
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4867..4915
                     /note="synthetic polyadenylation signal"
     rep_origin      5010..5465
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5097..5116)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5307..5328
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5532..5551)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5651..5673
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5711..5729)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5797..5901
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5902..5970,5971..6762)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6120..6139)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6933..7521
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7422..7441
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctccagta cgtgattctt gatcccgagc tggagccagg ggcgggcctt
      361 gcgctttagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc
      421 gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag
      481 ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa
      541 atgcgggcca ggatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg
      601 cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa
      661 tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt
      721 gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa
      781 gatggccgct tcccggccct gctccagggg gctcaaaatg gaggacgcgg cgctcgggag
      841 agcgggcggg tgagtcaccc acacaaagga aaggggcctt tccgtcctca gccgtcgctt
      901 catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc tggagctttt
      961 ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg
     1021 agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg
     1081 ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt
     1141 ttcttccatt tcaggtgtcg tgaacacggg ctgcagaagt tggtcgtgag gcactgggca
     1201 ggtaagtatc aaggttacaa gacaggttta aggagaccaa tagaaactgg gcttgtcgag
     1261 acagagaaga ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc
     1321 ctttctctcc acaggtgtcc actcccagtt caattacagc tcttaaggct agagtactta
     1381 atacgactca ctataggcta gcctcgagat gaaaggattt gtggggctga aaaatgctgg
     1441 tgctacttgt tacatgaact ctgtgatcca gcaactctat atgatcccct ctatcaggaa
     1501 tggtattctt gccatagaag gcacaggtag tgatgtagat gatgatatgt ctggggatga
     1561 gaagcaggac aacgagagca atgttgatcc cagggatgat gtgtttggat atcctcaaca
     1621 atttgaagac aaaccaccac tgagtaaaac agaagatagg aaagagtaca atattggtgt
     1681 tctaagacac cttcaggtta tctttggcca tttagctgct tctcgactac agtactatgt
     1741 gcctagagga ttttggaaac agttcaggct ttggggtgag cctgtcaatc ttcgtgaaca
     1801 acatgatgct ttagagttct ttaattcttt ggtggatagt ttagatgaag ctttaaaagc
     1861 cttagggcat ccagctatgc taagtaaagt cttgggaggt tcctttgctg atcagaaaat
     1921 ttgccaaggc tgcccacata ggtacgaatg tgaagaatct tttacgactc tgaatgtaga
     1981 cattagaaac caccaaaatc ttctggattc tttggaacag tatgtcaaag gagacttact
     2041 agaaggagca aatgcttatc attgtgaaaa atgcaataaa aaggttgata ctgtaaagcg
     2101 cttgctaatt aaaaaattgc ctcctgttct tgctatccaa ctaaaacgat tcgattatga
     2161 ctgggaaaga gaatgtgcaa tcaagttcaa tgattacttt gaatttcctc gagagttaga
     2221 catggaacct tacacagtgg caggtgttgc taaactggag ggagataatg tcaacccaga
     2281 aagtcagctg atacaacaga atgagcagtc tgaaagcgag aaagcaggaa gcacaaaata
     2341 cagacttgtg ggtgtgcttg tgcacagtgg tcaagcaagt ggcggacatt actattctta
     2401 catcattcag aggaatggag gggatggtga aaaaaatcgt tggtataaat ttgatgatgg
     2461 agatgtaacg gaatgtaaaa tggatgacga tgaagaaatg aaaaaccagt gttttggggg
     2521 agagtacatg ggagaagtat ttgatcacat gatgaagcgc atgtcgtaca ggcgtcagaa
     2581 aagatggtgg aatgcatata tactttttta tgaacgaatg gacggtggat caggcggtat
     2641 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     2701 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     2761 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     2821 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     2881 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     2941 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     3001 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     3061 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     3121 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     3181 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     3241 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     3301 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaaggact acaaagacca
     3361 tgacggtgat tataaagatc atgacatcga ctacaaggat gacgatgaca agtgaggcgg
     3421 ccgctctagc ccaattccgc cccccccccc ctaacgttac tggccgaagc cgcttggaat
     3481 aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg
     3541 tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc
     3601 tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt
     3661 cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg
     3721 acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac
     3781 cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg
     3841 tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg
     3901 ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc
     3961 cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggg cattgaacaa
     4021 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4081 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4141 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     4201 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4261 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     4321 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4381 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     4441 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     4501 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     4561 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     4621 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     4681 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     4741 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     4801 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgat
     4861 ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaatcg
     4921 atagcgataa ggatccgggc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     4981 aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc
     5041 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     5101 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5161 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5221 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5281 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5341 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5401 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     5461 aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg
     5521 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     5581 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5641 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5701 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     5761 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5821 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5881 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5941 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6001 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     6061 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     6121 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     6181 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     6241 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     6301 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6361 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6421 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6481 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6541 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6601 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6661 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6721 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6781 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6841 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6901 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6961 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     7021 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     7081 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     7141 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     7201 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     7261 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     7321 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     7381 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7441 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7501 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7561 tgctggcctt ttgctcacat ggctcgacag atcccccaat tcaagcttcg tga
//
