LOCUS       sequence-142793-        7489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HO1 and EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7489)
  AUTHORS   Cinti A, De Giorgi M, Chisci E, Arena C, Galimberti G, Farina L, 
            Bugarin C, Rivolta I, Gaipa G, Smolenski RT, Cerrito MG, Lavitrano 
            M, Giovannoni R
  TITLE     Simultaneous Overexpression of Functional Human HO-1, E5NT and 
            ENTPD1 Protects Murine Fibroblasts against TNF-alpha-Induced Injury 
            In Vitro.
  JOURNAL   PLoS One. 2015 Oct 29;10(10):e0141933. doi: 
            10.1371/journal.pone.0141933. eCollection 2015.
  PUBMED    26513260
REFERENCE   2  (bases 1 to 7489)
  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: "PLoS One.";
            date: "2015-10-29"; volume: "10(10):e0141933. doi"; pages: " 
            10.1371/journal.pone.0141933. eCollection 2015"
COMMENT     Sequence Label: pCX-HO1-2A-EGFP
FEATURES             Location/Qualifiers
     source          1..7489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        15..394
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        397..672
                     /label=chicken beta-actin promoter
     intron          673..1681
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1605..1627
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             2611..2676
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             join(2692..2694,2695..2697,2698..3411)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2692..2694,2695..2697,2698..3408)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2737..2758)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2998..3017)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3345..3366
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3502..3521)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3567..3622
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3621..3640)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3981..3997)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3981..3997)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3994..4016)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4005..4021
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4029..4035,4036..4053,4054..4059))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4074..4095
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    4277..4398
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4314..4333)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4368..4387
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4405..4860)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4542..4563)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4754..4773
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4887..4991
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4993..5350
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4993..5184
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5016..5036)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5201..5336
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5385..6179
                     /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             5385..6179
                     /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(5439..5458)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6049..6068
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6367..6386)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6411..6458
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6787..7375
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7276..7295
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattat ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
       61 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      121 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      181 aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt aaactgccca
      241 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      301 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      361 gtacatctac gtattagtca tcgctattac catgggtcga ggtgagcccc acgttctgct
      421 tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt attttttaat
      481 tattttgtgc agcgatgggg gcgggggggg ggggggcgcg cgccaggcgg ggcggggcgg
      541 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
      601 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
      661 gcgcggcggg cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg cgccgcctcg
      721 cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc
      781 ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggctcgtttc ttttctgtgg
      841 ctgcgtgaaa gccttaaagg gctccgggag ggccctttgt gcggggggga gcggctcggg
      901 gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct gcccggcggc
      961 tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcgtgtgcgc gaggggagcg
     1021 cggccggggg cggtgccccg cggtgcgggg gggctgcgag gggaacaaag gctgcgtgcg
     1081 gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc tgtaaccccc
     1141 ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgtg
     1201 cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg
     1261 gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc ggccccggag
     1321 cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga
     1381 gagggcgcag ggacttcctt tgtcccaaat ctggcggagc cgaaatctgg gaggcgccgc
     1441 cgcaccccct ctagcgggcg cgggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg
     1501 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccatctccag cctcggggct
     1561 gccgcagggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1621 tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca
     1681 gctcctgggc aacgtgctgg ttgttgtgct gtctcatcat tttggcaaag aattcccacc
     1741 atggagcgtc cgcaacccga cagcatgccc caggatttgt cagaggccct gaaggaggcc
     1801 accaaggagg tgcacaccca ggcagagaat gctgagttca tgaggaactt tcagaagggc
     1861 caggtgaccc gagacggctt caagctggtg atggcctccc tgtaccacat ctatgtggcc
     1921 ctggaggagg agattgagcg caacaaggag agcccagtct tcgcccctgt ctacttccca
     1981 gaagagctgc accgcaaggc tgccctggag caggacctgg ccttctggta cgggccccgc
     2041 tggcaggagg tcatccccta cacaccagcc atgcagcgct atgtgaagcg gctccacgag
     2101 gtggggcgca cagagcccga gctgctggtg gcccacgcct acacccgcta cctgggtgac
     2161 ctgtctgggg gccaggtgct caaaaagatt gcccagaaag ccctggacct gcccagctct
     2221 ggcgagggcc tggccttctt caccttcccc aacattgcca gtgccaccaa gttcaagcag
     2281 ctctaccgct cccgcatgaa ctccctggag atgactcccg cagtcaggca gagggtgata
     2341 gaagaggcca agactgcgtt cctgctcaac atccagctct ttgaggagtt gcaggagctg
     2401 ctgacccatg acaccaagga ccagagcccc tcacgggcac cagggcttcg ccagcgggcc
     2461 agcaacaaag tgcaagattc tgcccccgtg gagactccca gagggaagcc cccactcaac
     2521 acccgctccc aggctccgct tctccgatgg gtccttacac tcagctttct ggtggcgaca
     2581 gttgctgtag ggctttatgc catggctagc gtgaaacaga ctttgaattt tgaccttctc
     2641 aagttggcgg gagacgtgga gtccaaccca gggcccggca gcggccttaa gatggtgagc
     2701 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2761 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     2821 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     2881 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     2941 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     3001 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     3061 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     3121 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     3181 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     3241 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3301 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3361 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aggactcaga
     3421 attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct
     3481 ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa
     3541 gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg
     3601 ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca
     3661 tcagaatgag tatttggttt agagtttggc aacatatgcc atatgctggc tgccatgaac
     3721 aaaggtggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc cattccttat
     3781 tccatagaaa agccttgact tgaggttaga ttttttttat attttgtttt gtgttatttt
     3841 tttctttaac atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct
     3901 cctgactact cccagtcata gctgtccctc ttctcttatg aagatccctc gacctgcagc
     3961 ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4021 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4081 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4141 tgccagcgga tccaccggat ctagataact gatcataatc agccatacca catttgtaga
     4201 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     4261 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4321 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4381 actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     4441 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     4501 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     4561 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     4621 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     4681 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     4741 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     4801 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     4861 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4921 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4981 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     5041 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     5101 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     5161 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     5221 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     5281 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     5341 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     5401 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     5461 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     5521 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     5581 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     5641 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     5701 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     5761 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     5821 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     5881 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     5941 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     6001 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     6061 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     6121 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     6181 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     6241 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     6301 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     6361 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     6421 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     6481 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     6541 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     6601 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     6661 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6721 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6781 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6841 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6901 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     6961 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     7021 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     7081 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     7141 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     7201 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7261 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7321 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7381 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     7441 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
