LOCUS       sequence-438517-        8219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of IRSp53 pLxIS mutant in mammalian cells by retroviral 
            transduction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8219)
  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 8219)
  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-IRSp53(pLxIS) S269A
FEATURES             Location/Qualifiers
     source          1..8219
                     /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    1842..2408
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1858..2410
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2002..2019)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2229..2248
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2422..2424,2425..2427,2428..3141)
                     /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(2467..2488)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2728..2747)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3075..3096
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3203..3717
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3879..3895
                     /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(3903..3909,3910..3927,3928..3933))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3948..3969
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4086..4103)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4257..4845)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4337..4356)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5016..5807,5808..5876))
                     /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     5639..5658
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5877..5981)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6049..6067
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6105..6127)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6227..6246
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6440..6462
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6590..6609
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             6809..7325
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    7389..7730
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             7797..8213
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     8143..8165
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     8181..8197
                     /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 tatggccaat agcaatggct ccatccttcc aagtgccctg
     1441 tcagcttcca agtccaacct ggtcatcgcc gatcccattc ctggagccaa gcccttgcca
     1501 gtgccacctg agctggcacc atttgtgggg cggatgtctg ctcaggagaa tgtacctgtc
     1561 atgaatggcg tcgcaggccc ggacagcgag gactacaacc cctgggctga ccgaaaggct
     1621 gcccagccca aatccctgtc tcccccgcag tctcaaagca agctgagcga ctcgtactcc
     1681 aacacactcc ccgtgcgcaa gagcgtgacg ccgaagaaca gctatgccac cactgagaac
     1741 aagacactgc cacgttcaag ctccatggca gctggcctag aacgtaattg ataagcggcc
     1801 gcgtttaaac gtcgacggta tcgataagct tgatatcgaa ttccgccccc ccccctaacg
     1861 ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca
     1921 ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga
     1981 gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga
     2041 aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca
     2101 ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag
     2161 atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa
     2221 gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac
     2281 cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga
     2341 ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac
     2401 gatgataata tggccacaac catggtgagc aagggcgagg agctgttcac cggggtggtg
     2461 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtctggcgag
     2521 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     2581 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     2641 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     2701 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     2761 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     2821 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     2881 atggccgaca agcagaagaa cggcatcaag gcgaacttca agatccgcca caacatcgag
     2941 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     3001 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     3061 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     3121 atggacgagc tgtacaagta atgaattaat taagaattcg ataaaataaa agattttatt
     3181 tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca agctagctta
     3241 agtaacgcca ttttgcaagg catggaaaat acataactga gaatagagaa gttcagatca
     3301 aggttaggaa cagagagaca gcagaatatg ggccaaacag gatatctgtg gtaagcagtt
     3361 cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtcccgc cctcagcagt
     3421 ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct
     3481 tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag
     3541 ctcaataaaa gagcccacaa cccctcactc ggcgcgccag tcctccgata gactgcgtcg
     3601 cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct
     3661 gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcatggg
     3721 taacagtttc ttgaagttgg agaacaacat tctgagggta ggagtcgaat attaagtaat
     3781 cctgactcaa ttagccactg ttttgaatcc acatactcca atactcctga aatagttcat
     3841 tatggacagc gcagaaagag ctggggagaa ttgtgaaatt gttatccgct cacaattcca
     3901 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3961 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4021 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4081 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4141 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4201 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4261 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4321 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4381 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4441 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4501 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4561 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4621 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4681 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4741 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4801 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4861 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4921 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4981 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5041 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5101 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5161 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5221 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5281 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5341 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5401 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5461 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5521 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5581 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5641 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5701 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5761 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5821 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5881 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5941 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6001 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     6061 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     6121 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     6181 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     6241 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     6301 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     6361 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     6421 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggcgca aggaatggtg
     6481 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     6541 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     6601 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     6661 cgtagaggcg attagtccaa tttgttaaag acaggatatc agtggtccag gctctagttt
     6721 tgactcaaca atatcaccag ctgaagccta tagagtacga gccatagata aaataaaaga
     6781 ttttatttag tctccagaaa aaggggggaa tgaaagaccc cacctgtagg tttggcaagc
     6841 tagcttaagt aacgccattt tgcaaggcat ggaaaataca taactgagaa tagagaagtt
     6901 cagatcaagg ttaggaacag agagacagca gaatatgggc caaacaggat atctgtggta
     6961 agcagttcct gccccggctc agggccaaga acagatggtc cccagatgcg gtcccgccct
     7021 cagcagtttc tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc
     7081 tgtgccttat ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct
     7141 ccccgagctc aataaaagag cccacaaccc ctcactcggc gcgccagtcc tccgatagac
     7201 tgcgtcgccc gggtacccgt attcccaata aagcctcttg ctgtttgcat ccgaatcgtg
     7261 gactcgctga tccttgggag ggtctcctca gattgattga ctgcccacct cgggggtctt
     7321 tcatttggag gttccaccga gatttggaga cccctgccca gggaccaccg acccccccgc
     7381 cgggaggtaa gctggccagc ggtcgtttcg tgtctgtctc tgtctttgtg cgtgtttgtg
     7441 ccggcatcta atgtttgcgc ctgcgtctgt actagttagc taactagctc tgtatctggc
     7501 ggacccgtgg tggaactgac gagttctgaa cacccggccg caaccctggg agacgtccca
     7561 gggactttgg gggccgtttt tgtggcccga cctgaggaag ggagtcgatg tggaatccga
     7621 ccccgtcagg atatgtggtt ctggtaggag acgagaacct aaaacagttc ccgcctccgt
     7681 ctgaattttt gctttcggtt tggaaccgaa gccgcgcgtc ttgtctgctg cagcgctgca
     7741 gcatcgttct gtgttgtctc tgtctgactg tgtttctgta tttgtctgaa aattagggcc
     7801 agactgttac cactccctta agtttgacct taggtcactg gaaagatgtc gagcggatcg
     7861 ctcacaacca gtcggtagat gtcaagaaga gacgttgggt taccttctgc tctgcagaat
     7921 ggccaacctt taacgtcgga tggccgcgag acggcacctt taaccgagac ctcatcaccc
     7981 aggttaagat caaggtcttt tcacctggcc cgcatggaca cccagaccag gtcccctaca
     8041 tcgtgacctg ggaagccttg gcttttgacc cccctccctg ggtcaagccc tttgtacacc
     8101 ctaagcctcc gcctcctctt cctccatccg ccccgtctct cccccttgaa cctcctcgtt
     8161 cgaccccgcc tcgatcctcc ctttatccag ccctcactcc ttctctaggc gccggaatt
//
