LOCUS       sequence-422640-        7080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of gene  in mammalian cells by retroviral transduction .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7080)
  AUTHORS   Devant P, Dong Y, Mintseris J, Ma W, Gygi SP, Wu H, Kagan JC
  TITLE     Structural insights into cytokine cleavage by inflammatory 
            caspase-4.
  JOURNAL   Nature. 2023 Dec;624(7991):451-459. doi: 10.1038/s41586-023-06751-9.
            Epub 2023 Nov 22.
  PUBMED    37993712
REFERENCE   2  (bases 1 to 7080)
  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: 
            "10.1038/s41586-023-06751-9"; journalName: "Nature"; date: 
            "2023-12"; volume: "624"; issue: "7991"; pages: "451-459"
COMMENT     Sequence Label: pMSCV-human-pro-IL-18_E192K_D193K
FEATURES             Location/Qualifiers
     source          1..7080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1436..1445
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1445..1474
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2054..2077
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2112..2678
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2128..2680
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2272..2289)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2499..2518
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     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
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     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"
     LTR             3473..3987
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4149..4165
                     /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(4173..4179,4180..4197,4198..4203))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4218..4239
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4356..4373)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4527..5115)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4607..4626)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5286..6077,6078..6146))
                     /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     5909..5928
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6147..6251)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6319..6337
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6375..6397)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6497..6516
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6710..6732
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6860..6879
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7079..7080,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatctctcga gatcgatgcg gccgcgccac
     1441 catggaacaa aaactcatct cagaagagga tctgatggct gctgaaccag tagaagacaa
     1501 ttgcatcaac tttgtggcaa tgaaatttat tgacaatacg ctttacttta tagctgaaga
     1561 tgatgaaaac ctggaatcag attactttgg caagcttgaa tctaaattat cagtcataag
     1621 aaatttgaat gaccaagttc tcttcattga ccaaggaaat cggcctctat ttgaagatat
     1681 gactgattct gactgtagag ataatgcacc ccggaccata tttattataa gtatgtataa
     1741 agatagccag cctagaggta tggctgtaac tatctctgtg aagtgtgaga aaatttcaac
     1801 tctctcctgt gagaacaaaa ttatttcctt taaggaaatg aatcctcctg ataacatcaa
     1861 ggatacaaaa agtgacatca tattctttca gagaagtgtc ccaggacatg ataataagat
     1921 gcaatttgaa tcttcatcat acgaaggata ctttctagct tgtgaaaaag agagagacct
     1981 ttttaaactc attttgaaaa aagaggatga attgggggat agatctataa tgttcactgt
     2041 tcaaaacaaa aaggactaca aggacgacga tgacaagtag gtcgacggta tcgataagct
     2101 tgatatcgaa ttccgccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     2161 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     2221 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     2281 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     2341 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     2401 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     2461 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     2521 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     2581 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     2641 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac catggtgagc
     2701 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2761 aacggccaca agttcagcgt gtctggcgag 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 gcgaacttca 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 atgaattaat
     3421 taagaattcg ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga
     3481 ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaat
     3541 acataactga gaatagagaa gttcagatca aggttaggaa cagagagaca gcagaatatg
     3601 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     3661 gtccccagat gcggtcccgc cctcagcagt ttctagagaa ccatcagatg tttccagggt
     3721 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
     3781 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc
     3841 ggcgcgccag tcctccgata gactgcgtcg cccgggtacc cgtgtatcca ataaaccctc
     3901 ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt
     3961 gactacccgt cagcgggggt ctttcatggg taacagtttc ttgaagttgg agaacaacat
     4021 tctgagggta ggagtcgaat attaagtaat cctgactcaa ttagccactg ttttgaatcc
     4081 acatactcca atactcctga aatagttcat tatggacagc gcagaaagag ctggggagaa
     4141 ttgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4201 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4261 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4321 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4381 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4441 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4501 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4561 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4621 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4681 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4741 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4801 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4861 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4921 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4981 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5041 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5101 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5161 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5221 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5281 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5341 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5401 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5461 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5521 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5581 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5641 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5701 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5761 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5821 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5881 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5941 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6001 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6061 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6121 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6181 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6241 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     6301 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     6361 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     6421 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     6481 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     6541 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
     6601 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     6661 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     6721 cgttgtaaaa cgacggcgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     6781 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     6841 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     6901 cgccggtgat gccggccacg atgcgtccgg cgtagaggcg attagtccaa tttgttaaag
     6961 acaggatatc agtggtccag gctctagttt tgactcaaca atatcaccag ctgaagccta
     7021 tagagtacga gccatagata aaataaaaga ttttatttag tctccagaaa aaggggggaa
//
