LOCUS       sequence-426925-        9908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Episomal plasmid - for expression of 2 proteins linked with EGFP and
            n-terminal split puromycin resistance and split intein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9908)
  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 9908)
  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-nTermPuroR-nTermIntein
FEATURES             Location/Qualifiers
     source          1..9908
                     /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(3079..3667)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3159..3178)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(5244..5312,5313..6104)
                     /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(5462..5481)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6117..6253
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6405..6476
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6481..6527)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6545..6593
                     /note="synthetic polyadenylation signal"
     enhancer        6674..6977
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6979..7181
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7132..7152
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    join(7319..7892,7893..7895,7896..7904)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    join(7972..8545,8546..8548,8549..8557)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             join(8581..8583,8584..8586,8587..9297)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(8887..8906)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             9307..9363
                     /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 ctgcgagcga
     2701 tctctcgatt tcgatcaaga cattccttta atggtctttt ctggacacca ctaggggtca
     2761 gaagtagttc atcaaacttt cttccctccc taatctcaat cgcgaactaa tttgcgctgc
     2821 tggcgcatga agaataggac ttacgcgtgt ctgggtacga gcgtcagcct gcaggtgcct
     2881 aacttaatta accagtcaag tcagctactt ggcgagatcg acttgtctgg gtttcgacta
     2941 cgctcagaat tgcgtcagtc aagttcgatc tggtccttgc tattgcaccc gttctccgat
     3001 tacgagtttc atttaaatca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3061 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3121 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3181 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3241 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3301 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3361 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3421 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3481 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3541 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3601 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3661 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3721 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3781 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3841 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3901 tgcatttaaa tttccgaact ctccaaggcc ctcgtcggaa aatcttcaaa cctttcgtcc
     3961 gatccatctt gcaggctacc tctcgaacga actatcgcaa gtctcttggc cggccgcatt
     4021 ttctttgacc caggatattt gcaaaagaca tattcaaact tccgcagaac actttatttc
     4081 acatatacat gcctcttata tcagggatgt gaaacagggt cttgaaaact gtctaaatct
     4141 aaaacaatgc taatgcaggt ttaaatttaa taaaataaaa tccaaaatct aacagccaag
     4201 tcaaatctgt atgttttaac atttaaaata ttttaaagac gtcttttccc aggattcaac
     4261 atgtgaaatc ttttctcagg gatacacgtg tgcctagatc ctcattgctt tagtttttta
     4321 cagaggaatg aatataaaaa gaaaatactt aaattttatc cctcttacct ctataatcat
     4381 acataggcat aattttttaa cctaggctcc agatagccat agaagaacca aacactttct
     4441 gcgtgtgtga gaataatcag agtgagattt tttcacaagt acctgatgag ggttgagaca
     4501 ggtagaaaaa gtgagagatc tctatttatt tagcaataat agagaaagca tttaagagaa
     4561 taaagcaatg gaaataagaa atttgtaaat ttccttctga taactagaaa tagaggatcc
     4621 agtttctttt ggttaaccta aattttattt cattttattg ttttatttta ttttatttta
     4681 ttttattttg tgtaatcgta gtttcagagt gttagagctg aaaggaagaa gtaggagaaa
     4741 catgcaaagt aaaagtataa cactttcctt actaaaccga catgggtttc caggtagggg
     4801 caggattcag gatgactgac agggccctta gggaacactg agaccctacg ctgacctcat
     4861 aaatgcttgc tacctttgct gttttaatta catcttttaa tagcaggaag cagaactctg
     4921 cacttcaaaa gtttttcctc acctgaggag ttaatttagt acaaggggaa aaagtacagg
     4981 gggatggggg cgcgcctggt gtaccgagaa cgatcctctc agtgcgagtc tcgacgatcc
     5041 atatcgttgc ttggcagtca gccagtcgga atccagcttg ggacccagga agtccaatcg
     5101 tcagatattg tactcaagcc tggtcacggc agcgtaccga tctgtttaaa cctagatatt
     5161 gatagtctga tcggtcaacg tataatcgag tcctagcttt tgcaaacatc tatcaagaga
     5221 caggatcagc aggaggcttt cgcatgagta ttcaacattt ccgtgtcgcc cttattccct
     5281 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5341 atgctgaaga tcagttgggt gcgcgagtgg gttacatcga actggatctc aacagcggta
     5401 agatccttga gagttttcgc cccgaagaac gctttccaat gatgagcact tttaaagttc
     5461 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5521 tacactattc tcagaatgac ttggttgagt attcaccagt cacagaaaag catcttacgg
     5581 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5641 ccaacttact tctgacaacg attggaggac cgaaggagct aaccgctttt ttgcacaaca
     5701 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     5761 acgacgagcg tgacaccacg atgcctgtag caatggcaac aaccttgcgt aaactattaa
     5821 ctggcgaact acttactcta gcttcccggc aacagttgat agactggatg gaggcggata
     5881 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     5941 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6001 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6061 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaaccgatt ctaggtgcat
     6121 tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga
     6181 ttcagcaacg gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga
     6241 aatttatcct taagatcccg aatcgtttaa actcgactct ggctctatcg aatctccgtc
     6301 gtttcgagct tacgcgaaca gccgtggcgc tcatttgctc gtcgggcatc gaatctcgtc
     6361 agctatcgtc agcttacctt tttggcagcg atcgcggctc ccgacatctt ggaccattag
     6421 ctccacaggt atcttcttcc ctctagtggt cataacagca gcttcagcta cctctcaatt
     6481 caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctatca atcgttgcgt
     6541 tacacacaca aaaaaccaac acacatccat cttcgatgga tagcgatttt attatctaac
     6601 tgctgatcga gtgtagccag atctagtaat caattacggg gtcattagtt catagcccat
     6661 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     6721 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     6781 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     6841 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     6901 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     6961 tcatcgctat taccatgctg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     7021 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     7081 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     7141 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga
     7201 tcagatcttt gtcgatccta ccatccactc gacacacccg ccagcggccg cgccgccacc
     7261 atgtagattg cgaactaaga attctaagga tccaatcttc tactgtcgta tgccataacc
     7321 cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg
     7381 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga
     7441 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa
     7501 tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa
     7561 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct
     7621 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg
     7681 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg
     7741 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca
     7801 catgctttac atgtgtttag tcgaggttaa aaaacgtcta ggccccccga accacgggga
     7861 cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggat tagtttgcgc
     7921 tgctggcgca tgaagaataa gctagcttaa tgccataaag cgaatacgat tcccctctcc
     7981 ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt
     8041 ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg
     8101 ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg
     8161 tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc
     8221 tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca
     8281 aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag
     8341 ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa
     8401 ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt
     8461 tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt
     8521 ttcctttgaa aaacacgatg ataagcttgc cacaaccccg ggagatgagg atcgtttcgc
     8581 atggttagca aaggagaaga gttgttcact ggggttgtgc ccatcctggt agagctggac
     8641 ggagacgtaa acggccacaa gttcagcgtt tctggcgagg gagagggaga cgccacatac
     8701 ggaaagttga ctctgaagtt catctgcacc acaggtaagc tgcccgtgcc gtggcccaca
     8761 ctggtgacaa ctctgaccta cggagtgcag tgtttctcaa ggtacccaga ccacatgaag
     8821 cagcacgact tcttcaagag tgcgatgccc gagggctacg tccaggagcg cactatcttc
     8881 ttcaaggacg acggcaacta caagacacgg gctgaagtca agttcgaggg agacaccctg
     8941 gtgaaccgaa tcgagctgaa gggtatcgac ttcaaagagg acgggaacat tcttggacac
     9001 aaactggagt ataactacaa ctcccacaac gtatacataa tggccgacaa acagaagaac
     9061 ggtatcaaag tcaacttcaa gatccgccac aatatcgagg atggcagtgt gcaactcgca
     9121 gaccactacc agcagaacac gcctattggg gacggccccg tgcttctccc agacaaccac
     9181 tacctgagca cacagtcagc tctgtccaag gaccccaacg aaaagcggga ccacatggtg
     9241 ttgcttgagt tcgtgaccgc agcaggtatc accttgggca tggacgagct ttacaagggc
     9301 tccggcgcca cgaacttttc tctgcttaag caggcgggtg acgttgagga gaaccccggt
     9361 cccatgacag aatataaacc aacagttcga ttggcaacaa gagatgatgt gcctcaagct
     9421 gtcagaacac ttgcagctgc ctttgcggat tatccagcaa cccggcatac tgttgatccc
     9481 gatcggcata tagaacgagt tacagaactc caggagttgt ttctgacaag agtgggcctg
     9541 gatattggaa aagtctgggt ggcagatgat ggtgcagctg tcgctgtgtg gactacccca
     9601 gaatctgtgt ctggttactg cttggatctg aaaacccagg ttcagacccc gcagggtatg
     9661 aaggaaattt ccaacatcca ggtcggtgat ctggtactga gcaacacggg ttacaacgaa
     9721 gttctgaacg tcttcccgaa atctaaaaaa aagtcttaca aaatcaccct ggaagatggc
     9781 aaggaaatca tctgttccga agaacacctg tttccgacgc agactggtga aatgaacatc
     9841 tccggtggtc tgaaagaagg tatgtgtctg tatgttaaag aataagtcga cctatcaaat
     9901 atttaaag
//
