LOCUS       sequence-356644-        8521 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter plasmid utilizing d2eGFP as a reporter gene. Expression is 
            designed to mimick IFNB1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8521)
  AUTHORS   Thomsen EA, Andersen S, Marqvorsen MHS, Skipper KA, Paludan SR, 
            Mikkelsen JG
  TITLE     Single-Cell Monitoring of Activated Innate Immune Signaling by a 
            d2eGFP-Based Reporter Mimicking Time-Restricted Activation of IFNB1 
            Expression.
  JOURNAL   Front Cell Infect Microbiol. 2022 Jan 18;11:784762. doi: 
            10.3389/fcimb.2021.784762. eCollection 2021.
  PUBMED    35118008
REFERENCE   2  (bases 1 to 8521)
  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: "Front Cell 
            Infect Microbiol."; date: "2022-01-18"; volume: "11:784762. doi"; 
            pages: " 10.3389/fcimb.2021.784762. eCollection 2021"
COMMENT     Sequence Label: pCCL/IFNB1-d2eGFP-3'UTR
FEATURES             Location/Qualifiers
     source          1..8521
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     309..328
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     562..579
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    696..717
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(732..737,738..755,756..762)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    770..786
                     /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)."
     primer_bind     775..797
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     794..810
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     794..810
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     829..849
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        831..849
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     enhancer        950..1329
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1330..1533
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1484..1504
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             1548..1728
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1775..1900
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2393..2626
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2811..2855
                     /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    3122..3239
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             join(4270..4272,4273..4275,4276..4986,4987..4992,
                     4993..5115)
                     /codon_start=1
                     /product="EGFP destabilized by fusion to residues 422-461 
                     of mouse ornithine decarboxylase, giving an in vivo 
                     half-life of ~2 hours"
                     /label=d2EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKKLSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV
                     "
     primer_bind     complement(4315..4336)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4576..4595)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4923..4944
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5334..5922
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5387..5407)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5805..5816)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6011..6244
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(6273..6291)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(6274..6291)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(6537..6556)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6538..6556)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6566..6583)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6566..6582)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6575..6597)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6724..7179
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6811..6830)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7021..7042
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7205..7309
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7310..7378,7379..8170)
                     /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(7528..7547)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(8341..8521,1..408)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
       61 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      121 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      181 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      241 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      301 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      361 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
      421 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
      481 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
      541 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
      601 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
      661 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
      721 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
      781 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct
      841 cactaaaggg aacaaaagct ggagctgcaa gcttggccat tgcatacgtt gtatccatat
      901 cataatatgt acatttatat tggctcatgt ccaacattac cgccatgttg acattgatta
      961 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     1021 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     1081 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     1141 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     1201 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     1261 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     1321 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
     1381 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     1441 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
     1501 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgggg tctctctggt
     1561 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     1621 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     1681 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
     1741 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
     1801 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
     1861 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
     1921 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
     1981 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
     2041 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
     2101 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
     2161 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
     2221 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
     2281 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     2341 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     2401 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     2461 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     2521 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     2581 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     2641 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     2701 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     2761 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     2821 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     2881 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     2941 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     3001 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     3061 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcggttaa
     3121 cttttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     3181 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaatttta
     3241 tcgatcacga gactagcctc gacgatggtc tgccttctga gttctccatc ccaccctggt
     3301 tgggccttct ctatccttgt ctgtgttgtt tatatcctgc tgaagtgtga ttccacttgt
     3361 ggcagtttct cctctgtgta ggatcaaaag ggctgtggct ggttggtttg aaaatttctt
     3421 ataccctaga ctattccagt gcctttcaga agtttccaag gccctctcac actaatctat
     3481 tatcatattg ggcaaaactc cttgcagttt cagctactat tccctgattg acttttcagt
     3541 aaatctatct ctcagtcttt cagtatccaa agaagattgg ttctaggacc accatcccgc
     3601 tgcctccaca gataccaaaa tcagaggatg ctcaattccc tcttataaaa cgttgcagta
     3661 tttgcatata atctgcacat gtatttctgt atattttaaa tcatccctag attacttata
     3721 atacctgata caatataaat gctaaatagc tgtaacactg taactttaaa atttacatta
     3781 ttttttgttg ttgtattatt atttttattg tatttttaaa aaatattttc catctacagt
     3841 cagtagaatc cacggataca gaacctatgg ataggaagga ccaactgtat cttttagtgt
     3901 tttgaggttc ttgaattctc aggtcgtttg ctttcctttg ctttctccca agtcttgttt
     3961 tacaatttgc tttagtcatt cactgaaact ttaaaaaaca ttagaaaacc tcacagtttg
     4021 taaatctttt tccctattat atatatcata agataggagc ttaaataaag agttttagaa
     4081 actactaaaa tgtaaatgac ataggaaaac tgaaagggag aagtgaaagt gggaaattcc
     4141 tctgaataga gagaggacca tctcatataa ataggccata cccatggaga aaggacattc
     4201 taactacaac ctttcgaagc ctttgctctg gcacaacagg tagtaggcga cactgttcgt
     4261 gttgtcaaca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     4321 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ctggcgaggg cgagggcgat
     4381 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     4441 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     4501 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     4561 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     4621 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     4681 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     4741 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     4801 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     4861 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     4921 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     4981 tacaagaagc ttagccatgg cttcccgccg gaggtggagg agcaggatga tggcacgctg
     5041 cccatgtctt gtgcccagga gagcgggatg gaccgtcacc ctgcagcctg tgcttctgct
     5101 aggatcaatg tgtagagatc tcctagcctg tgcctctggg actggacaat tgcttcaagc
     5161 attcttcaac cagcagatgc tgtttaagtg actgatggct aatgtactgc atatgaaagg
     5221 acactagaag attttgaaat ttttattaaa ttatgagtta tttttattta tttaaatttt
     5281 attttggaaa ataaattatt tttggtgcaa aagtcactcg agggaattcc gataatcaac
     5341 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     5401 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     5461 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     5521 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     5581 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     5641 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     5701 ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg
     5761 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     5821 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     5881 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgggaa ttcgagctcg
     5941 gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     6001 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt
     6061 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     6121 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     6181 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     6241 agcagcatct agctagaatt aattccgtgt attctatagt gtcacctaaa tcgtatgtgt
     6301 atgatacata aggttatgta ttaattgtag ccgcgttcta acgacaatat gtacaagcct
     6361 aattgtgtag catctggctt actgaagcag accctatcat ctctctcgta aactgccgtc
     6421 agagtcggtt tggttggacg aaccttctga gtttctggta acgccgtccc gcacccggaa
     6481 atggtcagcg aaccaatcag cagggtcatc gctagcctag ggacgtaccc aattcgccct
     6541 atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     6601 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     6661 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     6721 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     6781 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     6841 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     6901 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     6961 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     7021 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     7081 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     7141 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     7201 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     7261 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     7321 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     7381 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     7441 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     7501 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     7561 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     7621 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     7681 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     7741 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     7801 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     7861 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     7921 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     7981 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     8041 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     8101 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     8161 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     8221 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     8281 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     8341 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     8401 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     8461 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     8521 c
//
