LOCUS       sequence-437123-        8200 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian Expression of Venus-iLID-CAAX.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8200)
  AUTHORS   Guntas G, Hallett RA, Zimmerman SP, Williams T, Yumerefendi H, Bear 
            JE, Kuhlman B
  TITLE     Engineering an improved light-induced dimer (iLID) for controlling 
            the localization and activity of signaling proteins.
  JOURNAL   Proc Natl Acad Sci U S A. 2015 Jan 6;112(1):112-7. doi: 
            10.1073/pnas.1417910112. Epub 2014 Dec 22.
  PUBMED    25535392
REFERENCE   2  (bases 1 to 8200)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.1417910112"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2015-01-6- 6"; volume: "112"; issue: "1"; pages: "112-7"
COMMENT     Sequence Label: pLL7.0: Venus-iLID-CAAX (from KRas4B)
FEATURES             Location/Qualifiers
     source          1..8200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2616..2928
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2847..2867
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     2945..2961
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3003..3036)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        3152..3455
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3456..3659
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      3730..3739
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3736..3738,3739..3741,3742..4452)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFDGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3781..3802)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4042..4061)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4389..4410
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4474..4911
                     /codon_start=1
                     /product="improved light-inducible dimer when used with 
                     SspB nano or SspB micro (Guntas et al., 2015)"
                     /label=iLID
                     /translation="LATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGR
                     NCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNVFHLQPMRDYKGDVQ
                     YFIGVQLDGTERLHGAAEREAVCLIKKTAFQIAEAANDENYF"
     protein_bind    complement(5002..5035)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5091..5677
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5144..5164)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5562..5573)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5680..5696)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             6206..6386
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6448..7036)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6528..6547)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7207..7998,7999..8067))
                     /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     7830..7849
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8068..8172)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgcgct ctagagatcc gacgcgccat ctctaggccc
     2641 gcgccggccc cctcgcacag acttgtggga gaagctcggc tactcccctg ccccggttaa
     2701 tttgcatata atatttccta gtaactatag aggcttaatg tgcgataaaa gacagataat
     2761 ctgttctttt taatactagc tacattttac atgataggct tggatttcta taagagatac
     2821 aaatactaaa ttattatttt aaaaaacagc acaaaaggaa actcacccta actgtaaagt
     2881 aattgtgtgt tttgagacta taaatatccc ttggagaaaa gccttgttaa cgcgcggtga
     2941 ccctcgaggt cgacggtatc gataagctcg cttcacgaga ttccagcagg tcgagggacc
     3001 taataacttc gtatagcata cattatacga agttatatta agggttccaa gcttaagcgg
     3061 ccgcgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt
     3121 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     3181 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     3241 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     3301 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     3361 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     3421 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     3481 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     3541 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     3601 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
     3661 gtttagtgaa ccgtcagatc cgctagaatt ctgcagtcga cggtaccgcg ggcccgggat
     3721 ccaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     3781 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     3841 ggcgatgcca cctacggcaa gctgaccctg aagttgatct gcaccaccgg caagctgccc
     3901 gtgccctggc ccaccctcgt gaccaccctc ggctacggcc tgcagtgctt cgcccgctac
     3961 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     4021 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     4081 gatggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     4141 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcaccgcc
     4201 gacaagcaga agaacggcat caaggcaaac ttcaagatcc gccacaacat cgaggacggc
     4261 ggcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     4321 ctgcccgaca accactacct gagctaccag tccaaactga gcaaagaccc caacgagaag
     4381 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     4441 gagctgtaca agggtagtgg tagtggggag tttctggcaa ccacactgga acggatcgag
     4501 aaaaatttcg tgattactga tccgagactg cctgacaacc caatcatttt tgcgagcgat
     4561 tccttcctgc agctgacaga atattctcgg gaagagatcc tggggcgcaa ttgccgtttt
     4621 ctgcagggac ccgagacaga ccgtgccact gttcggaaaa tcagagatgc tattgacaac
     4681 cagactgaag tgaccgttca gctgatcaat tataccaaga gcggcaagaa gttctggaac
     4741 gtgttccacc tgcagccgat gcgcgattat aagggcgacg tccagtactt cattggcgtg
     4801 cagctggatg gcaccgaacg tcttcatggc gccgctgagc gtgaggcggt ctgcctgatc
     4861 aaaaagacag cctttcagat tgctgaggca gcgaacgacg aaaattactt tggaagcggg
     4921 agtgggagca aaaagaagaa aaagaagtca aagacaaagt gtgtaattat gtagcctgca
     4981 ggcaattcgt cgagggacct aataacttcg tatagcatac attatacgaa gttatacatg
     5041 tttaagggtt ccggttccac taggtacaat tcgatatcaa gcttatcgat aatcaacctc
     5101 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     5161 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     5221 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     5281 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     5341 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     5401 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     5461 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     5521 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     5581 accttccttc ccggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     5641 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg acctcgatcg
     5701 agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg
     5761 tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc
     5821 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     5881 gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta
     5941 ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag ggatcagata
     6001 tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga aggtagaaga
     6061 agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg ggatggatga
     6121 cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc atcacatggc
     6181 ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct gagcctggga
     6241 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     6301 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     6361 ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa
     6421 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     6481 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6541 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6601 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6661 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6721 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6781 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6841 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6901 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6961 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     7021 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     7081 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     7141 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     7201 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     7261 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     7321 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     7381 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     7441 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     7501 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     7561 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7621 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7681 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7741 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7801 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7861 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7921 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7981 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     8041 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     8101 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     8161 aggggttccg cgcacatttc cccgaaaagt gccacctgac
//
