LOCUS       sequence-426927-        9925 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Episomal plasmid for expression of 2 proteins with puromycin 
            selection and EGFP monitoring.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9925)
  AUTHORS   Fallahee I, Hawiger D
  TITLE     Episomal Vectors for Stable Production of Recombinant Proteins and 
            Engineered Antibodies.
  JOURNAL   Antibodies (Basel). 2024 Mar 11;13(1):18. doi: 
            10.3390/antib13010018.
  PUBMED    38534208
REFERENCE   2  (bases 1 to 9925)
  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: "Antibodies 
            (Basel)."; date: "2024-03-11"; volume: "13(1):18. doi"; pages: " 
            10.3390/antib13010018"
COMMENT     Sequence Label: 
            pCMV-protein1-IRES-protein2-IRESatt-EGFP-2A-PuroR-SMAR-g5
FEATURES             Location/Qualifiers
     source          1..9925
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    2228..2349
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2239..2258)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2293..2312
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      2464..2510
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2540..2676
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2721..2792
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(3018..3606)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3098..3117)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(5183..5251,5252..6043)
                     /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(5401..5420)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6056..6192
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6344..6415
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6420..6466)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6484..6532
                     /note="synthetic polyadenylation signal"
     enhancer        6613..6916
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6918..7120
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7071..7091
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    join(7258..7831,7832..7834,7835..7843)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    join(7911..8484,8485..8487,8488..8496)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             join(8520..8522,8523..8525,8526..9236)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(8826..8845)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             9246..9302
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
ORIGIN
        1 aaaaaaaaat tgtatcaact ttctacaatc tctttcagaa gacagaagca gagggaatac
       61 ttcctaaatc attcaactag gccagcatta ccttaatacc ggaactagaa aatgacatta
      121 caagaaaaga aaacaacaga ccaatatctc tcatgaacaa agatacaaac attttcaaca
      181 aaatattagc aaaaagaatc caagaatgta tcaaaaaata tacaccacaa ccaagtagaa
      241 tttattccag atatgtaagg gtggttcaac gtttgaaaat caattaacgt aatttgtccc
      301 atcaacaggt taaagaagaa aatcacatgg tcatattgat agacacagaa aaagcatttg
      361 acaaaattta acacccattc atgatgcaat ctctcagtaa actaggaata gaggaaaact
      421 tcctcagctt gaatgtacct tcctctcaat tttgctatga acctgaaact cctcttaaaa
      481 aataaagttt ttcatttaaa aagaaaacaa aaaacatgga ggagcgttga tgtatctcat
      541 tttagaccaa tcagctatgg atagttaggc gacagcacag atagctgttg tacttctgtt
      601 tctggcaatg ttccagacta catttaaaaa atttttaatt atagacttgt acttaatgtt
      661 caagaaaaat atgaaaatgg ctttgccgtg ttaatgctac tcttttttaa aaaaaactaa
      721 agttcaaact ttatttatat ttcattagtt ttttagctac tgttcttttt ctgttctggg
      781 atctcattca gaatgccaca ttacatataa ttctcatgtc tccttgggtt cctcttagtt
      841 ttgacagttc ctcagacttt tcttattttt gatgaccttg acagttttga ggagtactgg
      901 ttagatatag ggtaatggtt tttaaagtat atttgtcatg atttatactg gggtaagggt
      961 ttggggagga agcccatggg gtaaagtact gttctcatca catcatatca aggttatata
     1021 ccatcaatat tgccacagat gttacttagc cttttaatat ttctctaatt tagtgtatat
     1081 gcaatgatag ttctctgatt tctgagattg agtttctcat gtgtaatgat tatttagagt
     1141 ttctctttca tctgttcaaa tttttgtcta gttttatttt ttactgattt gtaagacttc
     1201 tttttataat ctgcatatta caattctctt tactggggtg ttgcaaatat tttctgtcat
     1261 tctatggcct gacttttctt aatggttttt taattttaaa aataagtctt aatattcatg
     1321 caatctaatt aacaatcttt tctttgtggt taggactttg agtcataaga aatttttctc
     1381 tacactgaag tcatgatggc atgcttctat attattttct aaaagattta aagttttgcc
     1441 ttctccattt agacttataa ttcactggaa tttttttgtg tgtatggtat gacatatggg
     1501 ttccctttta ttttttacat ataaatatat ttccctgttt ttctaaaaaa gaaaaagatc
     1561 atcattttcc cattgtaaaa tgccatattt ttttcatagg tcacttacat atatcaatgg
     1621 gtctgtttct gagctctact ctattttatc agcctcactg tctatcccca cacatctcat
     1681 gctttgctct aaatcttgat atttagtgga acattctttc ccattttgtt ctacaagaat
     1741 atttttgtta ttgtcttttg ggcttctata tacattttag aatgaggttg gcaagttaac
     1801 aaacagcttt tttggggtga acatattgac tacaaattta tgtggaaaga aagtatacct
     1861 tcacaatatt aagtctttta gttcatgaat atagtatgtc tctccgtttc tgcattaact
     1921 tagacattca ttaatttctc tcacaattta taagtttatt tagatcttca ttcatttaaa
     1981 tcttcactaa cctctcattt acaatttgta agttttctgg gtaacagtct tgcacttctt
     2041 tgcctagatt tatttccaag tagattattt tcatacatcg tctatggtgt catttttaaa
     2101 atgtaatttt tcaccttttt attgctaaag agagatgact gattgttaat attgatcttg
     2161 tgcgtggcga ccttgctgtc tagattgact gactgagata cagcgtacct tcagctcaca
     2221 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     2281 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     2341 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     2401 gaggtttttt aaagcaagta aaacctctac aaatgtggta ttggcccatc tctatcggta
     2461 tcgtagcata accccttggg gcctctaaac gggtcttgag gggttttttg tgcccctcgg
     2521 gccggattgc tatctaccgg cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt
     2581 cttatactcc cacatatgcc agattcagca acggatacgg cttccccaac ttgcccactt
     2641 ccatacgtgt cctccttacc agaaatttat ccttaaggtc gtcagctatc ctgcaggcga
     2701 tctctcgatt tcgatcaaga cattccttta atggtctttt ctggacacca ctaggggtca
     2761 gaagtagttc atcaaacttt cttccctccc taatctcatt ggttaccttg ggctatcgaa
     2821 acttaattaa ccagtcaagt cagctacttg gcgagatcga cttgtctggg tttcgactac
     2881 gctcagaatt gcgtcagtca agttcgatct ggtccttgct attgcacccg ttctccgatt
     2941 acgagtttca tttaaatcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3001 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3061 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3121 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3181 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3241 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3301 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3361 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3421 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3481 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3541 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3601 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3661 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3721 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3781 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3841 gcatttaaat ttccgaactc tccaaggccc tcgtcggaaa atcttcaaac ctttcgtccg
     3901 atccatcttg caggctacct ctcgaacgaa ctatcgcaag tctcttggcc ggccgcattt
     3961 tctttgaccc aggatatttg caaaagacat attcaaactt ccgcagaaca ctttatttca
     4021 catatacatg cctcttatat cagggatgtg aaacagggtc ttgaaaactg tctaaatcta
     4081 aaacaatgct aatgcaggtt taaatttaat aaaataaaat ccaaaatcta acagccaagt
     4141 caaatctgta tgttttaaca tttaaaatat tttaaagacg tcttttccca ggattcaaca
     4201 tgtgaaatct tttctcaggg atacacgtgt gcctagatcc tcattgcttt agttttttac
     4261 agaggaatga atataaaaag aaaatactta aattttatcc ctcttacctc tataatcata
     4321 cataggcata attttttaac ctaggctcca gatagccata gaagaaccaa acactttctg
     4381 cgtgtgtgag aataatcaga gtgagatttt ttcacaagta cctgatgagg gttgagacag
     4441 gtagaaaaag tgagagatct ctatttattt agcaataata gagaaagcat ttaagagaat
     4501 aaagcaatgg aaataagaaa tttgtaaatt tccttctgat aactagaaat agaggatcca
     4561 gtttcttttg gttaacctaa attttatttc attttattgt tttattttat tttattttat
     4621 tttattttgt gtaatcgtag tttcagagtg ttagagctga aaggaagaag taggagaaac
     4681 atgcaaagta aaagtataac actttcctta ctaaaccgac atgggtttcc aggtaggggc
     4741 aggattcagg atgactgaca gggcccttag ggaacactga gaccctacgc tgacctcata
     4801 aatgcttgct acctttgctg ttttaattac atcttttaat agcaggaagc agaactctgc
     4861 acttcaaaag tttttcctca cctgaggagt taatttagta caaggggaaa aagtacaggg
     4921 ggatgggggc gcgcctggtg taccgagaac gatcctctca gtgcgagtct cgacgatcca
     4981 tatcgttgct tggcagtcag ccagtcggaa tccagcttgg gacccaggaa gtccaatcgt
     5041 cagatattgt actcaagcct ggtcacggca gcgtaccgat ctgtttaaac ctagatattg
     5101 atagtctgat cggtcaacgt ataatcgagt cctagctttt gcaaacatct atcaagagac
     5161 aggatcagca ggaggctttc gcatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5221 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5281 tgctgaagat cagttgggtg cgcgagtggg ttacatcgaa ctggatctca acagcggtaa
     5341 gatccttgag agttttcgcc ccgaagaacg ctttccaatg atgagcactt ttaaagttct
     5401 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5461 acactattct cagaatgact tggttgagta ttcaccagtc acagaaaagc atcttacgga
     5521 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5581 caacttactt ctgacaacga ttggaggacc gaaggagcta accgcttttt tgcacaacat
     5641 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5701 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca accttgcgta aactattaac
     5761 tggcgaacta cttactctag cttcccggca acagttgata gactggatgg aggcggataa
     5821 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5881 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5941 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6001 acagatcgct gagataggtg cctcactgat taagcattgg taaccgattc taggtgcatt
     6061 ggcgcagaaa aaaatgcctg atgcgacgct gcgcgtctta tactcccaca tatgccagat
     6121 tcagcaacgg atacggcttc cccaacttgc ccacttccat acgtgtcctc cttaccagaa
     6181 atttatcctt aagatcccga atcgtttaaa ctcgactctg gctctatcga atctccgtcg
     6241 tttcgagctt acgcgaacag ccgtggcgct catttgctcg tcgggcatcg aatctcgtca
     6301 gctatcgtca gcttaccttt ttggcagcga tcgcggctcc cgacatcttg gaccattagc
     6361 tccacaggta tcttcttccc tctagtggtc ataacagcag cttcagctac ctctcaattc
     6421 aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt atgctatcaa tcgttgcgtt
     6481 acacacacaa aaaaccaaca cacatccatc ttcgatggat agcgatttta ttatctaact
     6541 gctgatcgag tgtagccaga tctagtaatc aattacgggg tcattagttc atagcccata
     6601 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     6661 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     6721 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     6781 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     6841 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     6901 catcgctatt accatgctga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     6961 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     7021 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     7081 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
     7141 cagatctttg tcgatcctac catccactcg acacacccgc cagcggccgc gccgccacca
     7201 tgtagattgc gaactaagaa ttctaaggat ccaatcttct actgtcgtat gccataaccc
     7261 ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc
     7321 gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa
     7381 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat
     7441 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac
     7501 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg
     7561 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt
     7621 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg
     7681 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac
     7741 atgctttaca tgtgtttagt cgaggttaaa aaacgtctag gccccccgaa ccacggggac
     7801 gtggttttcc tttgaaaaac acgatgataa tatggccaca accatggatt agtttgcgct
     7861 gctggcgcat gaagaataag ctagcttaat gccataaagc gaatacgatt cccctctccc
     7921 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
     7981 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
     8041 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
     8101 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct
     8161 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
     8221 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
     8281 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag
     8341 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt
     8401 acatgtgttt agtcgaggtt aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     8461 tcctttgaaa aacacgatga taagcttgcc acaaccccgg gagatgagga tcgtttcgca
     8521 tggttagcaa aggagaagag ttgttcactg gggttgtgcc catcctggta gagctggacg
     8581 gagacgtaaa cggccacaag ttcagcgttt ctggcgaggg agagggagac gccacatacg
     8641 gaaagttgac tctgaagttc atctgcacca caggtaagct gcccgtgccg tggcccacac
     8701 tggtgacaac tctgacctac ggagtgcagt gtttctcaag gtacccagac cacatgaagc
     8761 agcacgactt cttcaagagt gcgatgcccg agggctacgt ccaggagcgc actatcttct
     8821 tcaaggacga cggcaactac aagacacggg ctgaagtcaa gttcgaggga gacaccctgg
     8881 tgaaccgaat cgagctgaag ggtatcgact tcaaagagga cgggaacatt cttggacaca
     8941 aactggagta taactacaac tcccacaacg tatacataat ggccgacaaa cagaagaacg
     9001 gtatcaaagt caacttcaag atccgccaca atatcgagga tggcagtgtg caactcgcag
     9061 accactacca gcagaacacg cctattgggg acggccccgt gcttctccca gacaaccact
     9121 acctgagcac acagtcagct ctgtccaagg accccaacga aaagcgggac cacatggtgt
     9181 tgcttgagtt cgtgaccgca gcaggtatca ccttgggcat ggacgagctt tacaagggat
     9241 caggagctac aaatttctca ctcttgaaac aagctgggga tgtagaagaa aatcctgggc
     9301 caatgacaga atataaacct actgttaggc tggcaacacg agatgatgtg ccaagagctg
     9361 tgagaacgtt ggctgctgct tttgctgatt atccagctac ccgacataca gtagacccag
     9421 ataggcatat agaaagggtg acagaactcc aggagctgtt tctgacacgt gtgggcttgg
     9481 atattgggaa agtctgggtg gcagatgatg gtgctgccgt tgccgtatgg actacccccg
     9541 aatctgttga ggccggcgcc gttttcgcag aaattggacc cagaatggct gaactttccg
     9601 gctcaagact tgctgctcaa cagcaaatgg aaggtctgct tgctccacat aggccaaaag
     9661 aaccagcttg gttcttggca acagttgggg tgagtcctga tcatcaaggt aaaggccttg
     9721 ggtcagctgt tgtcctgcct ggcgttgaag cggcagaaag agcgggtgtc ccagcatttc
     9781 ttgaaacgag cgccccaaga aatctgccat tttatgaaag attggggttt acagtaacag
     9841 cagatgttga agtacccgag ggcccccgta cgtggtgtat gactagaaaa ccaggagctt
     9901 aagtcgacct atcaaatatt taaag
//
