LOCUS       sequence-251593-        8298 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8298)
  AUTHORS   Meyer C, Garzia A, Morozov P, Molina H, Tuschl T
  TITLE     The G3BP1-Family-USP10 Deubiquitinase Complex Rescues Ubiquitinated 
            40S Subunits of Ribosomes Stalled in Translation from Lysosomal 
            Degradation.
  JOURNAL   Mol Cell. 2020 Mar 19;77(6):1193-1205.e5. doi: 
            10.1016/j.molcel.2019.12.024. Epub 2020 Jan 24.
  PUBMED    31981475
REFERENCE   2  (bases 1 to 8298)
  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.2019.12.024"; journalName: "Mol Cell"; date: 
            "2020-03-19- 19"; volume: "77"; issue: "6"; pages: "1193-1205.e5"
COMMENT     Sequence Label: pLENTI_RPS3_K230R
FEATURES             Location/Qualifiers
     source          1..8298
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        253..456
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     482..501
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        482..500
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1314..1343
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1362..1385
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1419..1992
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1442..1994
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1586..1603)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1813..1832
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1994..2593
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1994..2013)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2490..2510
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(2623..2656)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2712..3300
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2765..2785)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3183..3194)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3303..3319)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3304..3320)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3829..4009
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4071..4659)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4151..4170)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4736..5395)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5310..5329
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5396..5500)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5575..5594)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5765..6144
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6146..6344
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             6362..6542
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6589..6714
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7211..7444
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7629..7673
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    7971..8088
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        join(8171..8298,1..252)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
       61 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      121 catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      181 gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt attagtcatc
      241 gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac
      301 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      361 aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt
      421 aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagaatttt
      481 gtaatacgac tcactatagg gcggccggga attcgtcgac tggatccggt accgaggaga
      541 tctgccgccg cgatcgccgg cgcgccatgg cagtgcaaat atccaagaag aggaagtttg
      601 tcgctgatgg catcttcaaa gctgaactga atgagtttct tactcgggag ctggctgaag
      661 atggctactc tggagttgag gtgcgagtta caccaaccag gacagaaatc attatcttag
      721 ccaccagaac acagaatgtt cttggtgaga agggccggcg gattcgggaa ctgactgctg
      781 tagttcagaa gaggtttggc tttccagagg gcagtgtaga gctttatgct gaaaaggtgg
      841 ccactagagg tctgtgtgcc attgcccagg cagagtctct gcgttacaaa ctcctaggag
      901 ggcttgctgt gcggagggcc tgctatggtg tgctgcggtt catcatggag agtggggcca
      961 aaggctgcga ggttgtggtg tctgggaaac tccgaggaca gagggctaaa tccatgaagt
     1021 ttgtggatgg cctgatgatc cacagcggag accctgttaa ctactacgtt gacactgctg
     1081 tgcgccacgt gttgctcaga cagggtgtgc tgggcatcaa ggtgaagatc atgctgccct
     1141 gggacccaac tggtaagatt ggccctaaga agcccctgcc tgaccacgtg agcattgtgg
     1201 aacccaaaga tgagatactg cccaccaccc ccatctcaga acagaagggt gggaggccag
     1261 agccgcctgc catgccccag ccagtcccca cagcaacgcg tacgcggccg ctcgagcaga
     1321 aactcatctc agaagaggat ctggcagcaa atgatatcct ggattacaag gatgacgacg
     1381 ataaggttta aacctaggcg tagcggccgc aaattccgcc cctctccctc ccccccccct
     1441 aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt
     1501 tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg
     1561 acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc
     1621 gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt
     1681 tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta
     1741 taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg
     1801 gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag
     1861 gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag
     1921 tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa
     1981 acacgatgat aatatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     2041 cccccgggca gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     2101 cgtcgatcca gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     2161 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     2221 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     2281 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     2341 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     2401 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     2461 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     2521 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     2581 gcccggtgcc tgatgtacaa gtaggattcg tcgagggacc taataacttc gtatagcata
     2641 cattatacga agttatacat gtttaagggt tccggttcca ctaggtacaa ttcgatatca
     2701 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     2761 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     2821 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     2881 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     2941 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     3001 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3061 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     3121 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     3181 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     3241 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     3301 atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac aagtagcaat
     3361 acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt
     3421 tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt
     3481 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     3541 gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac
     3601 acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca
     3661 gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct
     3721 gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc
     3781 cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg
     3841 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     3901 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     3961 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag catgtgagca
     4021 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4081 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4141 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4201 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4261 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4321 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4381 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4441 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4501 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4561 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4621 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4681 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgattacg
     4741 ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga
     4801 agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt
     4861 gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt
     4921 ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa
     4981 taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata
     5041 tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag
     5101 tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt
     5161 ctttcattgc catacggaac tccggatgag cattcatcag gcgggcaaga atgtgaataa
     5221 aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca
     5281 gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt
     5341 tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc tccatactct
     5401 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5461 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5521 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     5581 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     5641 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     5701 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatcg
     5761 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     5821 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     5881 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     5941 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     6001 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     6061 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     6121 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     6181 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     6241 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     6301 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt ttgcctgtac
     6361 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     6421 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     6481 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     6541 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
     6601 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
     6661 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
     6721 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
     6781 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
     6841 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     6901 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     6961 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     7021 caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc gctgatcttc agacctggag
     7081 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg
     7141 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag
     7201 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg
     7261 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc
     7321 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg
     7381 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc
     7441 tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg
     7501 ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg
     7561 acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc
     7621 agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt
     7681 ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct
     7741 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat
     7801 attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag
     7861 gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat
     7921 cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa ttttaaaaga
     7981 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
     8041 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
     8101 agggacagca gagatccagt ttggttagta ccgggcccgc tctagacatg tccaatatga
     8161 ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     8221 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     8281 tgaccgccca acgacccc
//
