LOCUS       sequence-328407-        6189 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Modified LIC 6D backbone with a C-terminal TEV-GFP-FLAG tag. Derived
            from Scott Gradia's pcDNA3.1 LIC 6D by Liron David..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6189)
  AUTHORS   Hollingsworth LR, Sharif H, Griswold AR, Fontana P, Mintseris J, 
            Dagbay KB, Paulo JA, Gygi SP, Bachovchin DA, Wu H
  TITLE     DPP9 sequesters the C terminus of NLRP1 to repress inflammasome 
            activation.
  JOURNAL   Nature. 2021 Mar 17. pii: 10.1038/s41586-021-03350-4. doi: 
            10.1038/s41586-021-03350-4.
  PUBMED    33731932
REFERENCE   2  (bases 1 to 6189)
  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: "Nature. 
            2021 Mar 17. pii: 10.1038/s41586-021-03350-4. doi: 
            10.1038/s41586-021-03350-4."
COMMENT     Sequence Label: pcDNA3 TEV-GFP-FLAG LIC cloning vector (6LD)
FEATURES             Location/Qualifiers
     source          1..6189
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             944..964
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(983..985,986..1696)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1025..1046)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1633..1654
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1697..1720
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(1742..1760)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1743..1760)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1780..1797)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1786..2010
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2056..2484
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2143..2162)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2353..2374
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2493..2513)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2498..2827
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2678..2813
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2740..2759
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2894..3688
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2948..2967)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3558..3577
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3862..3983
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3899..3918)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3953..3972
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4032..4048)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4032..4048)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4045..4067)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4056..4072
                     /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(4080..4086,4087..4104,4105..4110))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4125..4146
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4263..4280)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4434..5022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4514..4533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5193..5984,5985..6053))
                     /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     5816..5835
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6054..6158)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccctacttcc aatccaatat tggtagtggg agcgaaaacc tttacttcca
      961 gggcaacggc agcagcggat ccgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1021 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgc gcggcgaggg
     1081 cgagggcgat gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1141 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1201 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1261 ccaggagcgc accatctcct tcaaggacga cggcacctac aagacccgcg ccgaggtgaa
     1321 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1381 cggcaacatc ctggggcaca agctggagta caacttcaac agccacaacg tctatatcac
     1441 ggccgacaag cagaagaacg gcatcaaggc gaacttcaag atccgccaca acgtcgagga
     1501 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1561 gctgctgccc gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga
     1621 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     1681 ggacgagctg tacaaggact acaaggacga tgacgacaag tagtaatcta gagggcccta
     1741 ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt
     1801 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     1861 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     1921 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     1981 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2041 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2101 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2161 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2221 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2281 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2341 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2401 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2461 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     2521 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2581 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     2641 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     2701 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     2761 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     2821 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     2881 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     2941 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     3001 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     3061 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     3121 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     3181 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     3241 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     3301 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     3361 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     3421 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     3481 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     3541 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     3601 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     3661 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     3721 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     3781 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     3841 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     3901 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3961 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     4021 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4081 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4141 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4201 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4261 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4321 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4381 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4441 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4501 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4561 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4621 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4681 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4741 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4801 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4861 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4921 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4981 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5041 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5101 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5161 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5221 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5281 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5341 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5401 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5461 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5521 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5581 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5641 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5701 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5761 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5821 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5881 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5941 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6001 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6061 ctttttcatt attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6121 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6181 cctgacgtc
//
