LOCUS       sequence-438527-        7988 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CNOT6 (pLxIS) in mammalian cells by retroviral 
            transduction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7988)
  AUTHORS   Landau LM, Chaudhary N, Tien YC, Rogozinska M, Joshi S, Yao C, 
            Crowley J, Hullahalli K, Campbell IW, Waldor MK, Haigis M, Kagan JC
  TITLE     pLxIS-containing domains are biochemically flexible regulators of 
            interferons and metabolism.
  JOURNAL   Mol Cell. 2024 Jul 11;84(13):2436-2454.e10. doi: 
            10.1016/j.molcel.2024.05.030. Epub 2024 Jun 25.
  PUBMED    38925114
REFERENCE   2  (bases 1 to 7988)
  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: 
            "10.1016/j.molcel.2024.05.030"; journalName: "Mol Cell"; date: 
            "2024-07-11- 11"; volume: "84"; issue: "13"; pages: "2436-2454.e10"
COMMENT     Sequence Label: pMSCV-FKBP-CNOT6
FEATURES             Location/Qualifiers
     source          1..7988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      7..16
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             16..81
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    1611..2177
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1627..2179
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1771..1788)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1998..2017
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2191..2193,2194..2196,2197..2910)
                     /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(2236..2257)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2497..2516)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2844..2865
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             2972..3486
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3648..3664
                     /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(3672..3678,3679..3696,3697..3702))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3717..3738
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3855..3872)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4026..4614)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4106..4125)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4785..5576,5577..5645))
                     /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     5408..5427
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5646..5750)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5818..5836
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5874..5896)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5996..6015
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6209..6231
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6359..6378
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             6578..7094
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    7158..7499
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             7566..7982
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     7912..7934
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     7950..7966
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
ORIGIN
        1 agatctgcca ccatggacta caaggatcac gacggtgatt acaaggacca cgacatcgac
       61 tacaaggacg acgacgacaa gggcgttcaa gtagaaacaa tatctccagg agacgggcgt
      121 acatttccta agcgtggcca aacttgcgta gtccattata ccggcatgct ggaggacggt
      181 aagaaggtcg atagctctag agaccgtaac aaacctttca aatttatgct ggggaaacag
      241 gaggttatcc gtggttggga ggaaggcgtg gcccaaatgt ccgttgggca gcgtgctaag
      301 ttaaccatct cgccagatta cgcatatggg gctacaggcc atcctgggat cattcccccg
      361 catgcgacac ttgtgtttga tgtagagctg ttgaagttgg agacgcgggg agttcaagtc
      421 gaaacgattt caccaggtga tggacgcacc tttcctaaac ggggacaaac gtgcgtcgtg
      481 cattacaccg gtatgctgga agacggcaag aaagtcgatt caagccgcga tagaaataaa
      541 cccttcaaat tcatgcttgg aaaacaggaa gtaatacgcg gatgggaaga gggtgtggcg
      601 caaatgtctg tcgggcaaag agctaaactg acaataagtc ctgattacgc gtacggtgcg
      661 actgggcacc ctggtatcat tccgccgcac gcgacacttg tcttcgacgt cgagttactt
      721 aagcttgaga cgcggggggt gcaggtagag actatttcgc ccggtgacgg aagaactttc
      781 ccaaagcggg gacagacctg tgttgtccac tacactggga tgttggagga tggtaagaaa
      841 gttgactcaa gtcgtgatcg caacaaaccc tttaagttta tgcttgggaa gcaggaagtc
      901 atcagaggtt gggaagaagg cgtagctcag atgtcagtgg gacaacgggc caaattaacc
      961 atcagtccag actacgccta cggagccacc ggtcacccag ggatcatacc acctcacgca
     1021 accttagtgt tcgatgtgga acttttaaag ttagagacgc gcggtgtaca agtggaaaca
     1081 atcagtccag gggatgggag aacattcccc aaaagaggcc agacgtgtgt cgtgcactat
     1141 acggggatgt tagaagatgg gaaaaaggtc gattcgtcac gcgaccgcaa caaaccgttt
     1201 aaattcatgt tgggaaagca ggaggtgatt cgcggctggg aggaaggtgt ggcacagatg
     1261 agcgtgggcc agagagcaaa actgactatc tctcctgatt atgcctacgg cgcgacgggc
     1321 caccctggca taattccacc ccacgctacc ctggtattcg atgtcgagct tctgaagctt
     1381 gagggtgctg gggctggggc tatgcccaaa gaaaagtacg agccccctga ccctcggagg
     1441 atgtacacaa ttatgtcctc tgaggaagca gcaaatggaa agaaatcaca ttgggcagag
     1501 cttgaaataa gtggaaaagt aagaagctta agctcatctt tgtggtcact cactcactga
     1561 taagcggccg cgtttaaacg tcgacggtat cgataagctt gatatcgaat tccgcccccc
     1621 cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt
     1681 tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct
     1741 tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga
     1801 atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga
     1861 ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac
     1921 gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag
     1981 ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc
     2041 agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg
     2101 tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt
     2161 gaaaaacacg atgataatat ggccacaacc atggtgagca agggcgagga gctgttcacc
     2221 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2281 tctggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     2341 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     2401 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2461 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2521 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2581 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     2641 gtctatatca tggccgacaa gcagaagaac ggcatcaagg cgaacttcaa gatccgccac
     2701 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     2761 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     2821 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2881 actctcggca tggacgagct gtacaagtaa tgaattaatt aagaattcga taaaataaaa
     2941 gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta ggtttggcaa
     3001 gctagcttaa gtaacgccat tttgcaaggc atggaaaata cataactgag aatagagaag
     3061 ttcagatcaa ggttaggaac agagagacag cagaatatgg gccaaacagg atatctgtgg
     3121 taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtcccgcc
     3181 ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac
     3241 cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg
     3301 ctccccgagc tcaataaaag agcccacaac ccctcactcg gcgcgccagt cctccgatag
     3361 actgcgtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt
     3421 ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc
     3481 tttcatgggt aacagtttct tgaagttgga gaacaacatt ctgagggtag gagtcgaata
     3541 ttaagtaatc ctgactcaat tagccactgt tttgaatcca catactccaa tactcctgaa
     3601 atagttcatt atggacagcg cagaaagagc tggggagaat tgtgaaattg ttatccgctc
     3661 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3721 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3781 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3841 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3901 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3961 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4021 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4081 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4141 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4201 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4261 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4321 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4381 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4441 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4501 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4561 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4621 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4681 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4741 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4801 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4861 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4921 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4981 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5041 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5101 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5161 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5221 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5281 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5341 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5401 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5461 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5521 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5581 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5641 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5701 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5761 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     5821 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     5881 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     5941 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     6001 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
     6061 aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg
     6121 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     6181 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggcgcaa
     6241 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     6301 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     6361 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     6421 tgcgtccggc gtagaggcga ttagtccaat ttgttaaaga caggatatca gtggtccagg
     6481 ctctagtttt gactcaacaa tatcaccagc tgaagcctat agagtacgag ccatagataa
     6541 aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt
     6601 ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaatacat aactgagaat
     6661 agagaagttc agatcaaggt taggaacaga gagacagcag aatatgggcc aaacaggata
     6721 tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg
     6781 tcccgccctc agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg
     6841 aaatgaccct gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc
     6901 gcttctgctc cccgagctca ataaaagagc ccacaacccc tcactcggcg cgccagtcct
     6961 ccgatagact gcgtcgcccg ggtacccgta ttcccaataa agcctcttgc tgtttgcatc
     7021 cgaatcgtgg actcgctgat ccttgggagg gtctcctcag attgattgac tgcccacctc
     7081 gggggtcttt catttggagg ttccaccgag atttggagac ccctgcccag ggaccaccga
     7141 cccccccgcc gggaggtaag ctggccagcg gtcgtttcgt gtctgtctct gtctttgtgc
     7201 gtgtttgtgc cggcatctaa tgtttgcgcc tgcgtctgta ctagttagct aactagctct
     7261 gtatctggcg gacccgtggt ggaactgacg agttctgaac acccggccgc aaccctggga
     7321 gacgtcccag ggactttggg ggccgttttt gtggcccgac ctgaggaagg gagtcgatgt
     7381 ggaatccgac cccgtcagga tatgtggttc tggtaggaga cgagaaccta aaacagttcc
     7441 cgcctccgtc tgaatttttg ctttcggttt ggaaccgaag ccgcgcgtct tgtctgctgc
     7501 agcgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaaa
     7561 attagggcca gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg
     7621 agcggatcgc tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct
     7681 ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc
     7741 tcatcaccca ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg
     7801 tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct
     7861 ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac
     7921 ctcctcgttc gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg
     7981 ccggaatt
//
