LOCUS       sequence-112292-        9181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of Tiam DH/PH-tgRFPt-Nano.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9181)
  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 9181)
  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: 
            "10.1073/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: mTiam1(64-437)-tgRFPt-SSPB WT
FEATURES             Location/Qualifiers
     source          1..9181
                     /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    1686..1919
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2104..2148
                     /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    2446..2563
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2618..2930
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2849..2869
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     2947..2963
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3005..3038)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        3154..3457
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3458..3661
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             4873..5601
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     CDS             5677..6006
                     /codon_start=1
                     /product="SspB from Haemophilus influenzae, with the 
                     mutations Y11K and A15E to disrupt the natural dimerization
                     of the protein"
                     /label=SspB nano
                     /note="dimerizes with iLID at high affinity to create iLID 
                     nano (Guntas et al., 2015)"
                     /translation="SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQ
                     IVLNLSASATGNLQLTNDFIQFNARFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     misc_feature    6068..6658
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6121..6141)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6539..6550)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6661..6677)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             7187..7367
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7429..8017)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7509..7528)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8188..8979,8980..9048))
                     /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     8811..8830
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9049..9153)
                     /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 gccgcgctga tcttcagacc tggaggagga
     1561 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     1621 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     1681 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     1741 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     1801 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1861 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1921 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1981 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     2041 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     2101 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     2161 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     2221 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     2281 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     2341 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     2401 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     2461 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2521 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2581 cagcagagat ccagtttggt tagtaccgcg ctctagagat ccgacgcgcc atctctaggc
     2641 ccgcgccggc cccctcgcac agacttgtgg gagaagctcg gctactcccc tgccccggtt
     2701 aatttgcata taatatttcc tagtaactat agaggcttaa tgtgcgataa aagacagata
     2761 atctgttctt tttaatacta gctacatttt acatgatagg cttggatttc tataagagat
     2821 acaaatacta aattattatt ttaaaaaaca gcacaaaagg aaactcaccc taactgtaaa
     2881 gtaattgtgt gttttgagac tataaatatc ccttggagaa aagccttgtt aacgcgcggt
     2941 gaccctcgag gtcgacggta tcgataagct cgcttcacga gattccagca ggtcgaggga
     3001 cctaataact tcgtatagca tacattatac gaagttatat taagggttcc aagcttaagc
     3061 ggccgcgtgg ataaccgtat taccgccatg cattagttat taatagtaat caattacggg
     3121 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     3181 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     3241 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     3301 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     3361 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     3421 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     3481 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     3541 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     3601 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     3661 tggtttagtg aaccgtcaga tccgctagaa ttctgcagtc gacggtaccg cgggcccgcc
     3721 accatgcgac agctgtcgga tgcggataag ctgcgcaagg tgatctgtga actgctggag
     3781 actgaacgca cctatgtgaa agacttaaac tgcctcatgg agagatacct gaagcccctt
     3841 cagaaggaga cctttctcac ccaggatgag cttgatgtac tttttggaaa tttaaccgaa
     3901 atggtggagt ttcaagtcga gttccttaag actctagaag atggagtaag actggtccct
     3961 gacttggaaa agctggagaa ggttgaccag ttcaagaaag tgctcttctc tctgggggga
     4021 tccttcctgt actacgcgga ccgcttcaag ctctacagtg ccttctgtgc gagccacaca
     4081 aaagtcccca aggtcctggt gaaagccaag acggacacag ccttcaaggc gttcctcgat
     4141 gcccagaacc cgaggcagca gcactcatcc acgctggagt cttacctcat caagcccatc
     4201 cagagggtcc tcaagtaccc gcttctgctc agggagctgt ttgcgctgac cgatgcggag
     4261 agcgaggaac actaccacct ggacgtggcc atcaagacta tgaacaaggt cgccagtcac
     4321 atcaatgaga tgcagaagat ccacgaagag tttggtgctg tgtttgacca gctgattgct
     4381 gagcagacgg gagagaagaa agaggttgca gatctgagca tgggtgacct gctcttgcac
     4441 accagcgtca tctggctgaa cccacccgcc tcactgggaa agtggaaaaa ggagccagaa
     4501 ttagcagcct tcgtcttcaa aacggccgtg gtccttgtat ataaagacgg ttccaagcag
     4561 aagaagaaac ttgttggctc tcacaggctg tcaatctacg aggagtggga ccctttccgg
     4621 tttcgccaca tgatccctac cgaagctttg caggtccgag ctctgcccag tgcagatgca
     4681 gaggcaaatg cggtatgcga aattgtccac gtgaaatcag agtcagaagg gaggcccgag
     4741 cgggttttcc acctctgctg cagttcccca gagagcagaa aggactttct gaagtctgtg
     4801 cattcgatcc tgcgagataa acacagaaga cagctcctca aaacggaaga attcggtagt
     4861 ggaagtggtt ccgtgtctaa gggcgaagag ctgattaagg agaacatgca catgaagctg
     4921 tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag
     4981 ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc tctccccttc
     5041 gccttcgaca tcctggctac cagcttcatg tacggcagca gaaccttcat caaccacacc
     5101 cagggcatcc ccgacttctt taagcagtcc ttccctgagg gcttcacatg ggagagagtc
     5161 accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc
     5221 tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg
     5281 cagaagaaaa cactcggctg ggaggccaac accgagatgc tgtaccccgc tgacggcggc
     5341 ctggaaggca gaaccgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac
     5401 ttcaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac
     5461 tatgtggacc acagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag
     5521 cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca caaacttaat
     5581 ggcatggacg agctgtacaa gtccggactc agatctcgag ctcaagcttc gaacgaattc
     5641 ggcattgatc tgagcggcct gaccctgcag gaattcagct ccccgaaacg ccctaagctg
     5701 ctgcgtgaat attacgattg gctggttgat aacagcttta ccccatatct ggtggtggat
     5761 gccacatacc tgggcgtgaa cgtgcccgtg gagtatgtga aagacggtca gatcgtgctg
     5821 aatctgtctg caagtgcgac cggcaacctg caactgacaa atgattttat ccagttcaac
     5881 gcccgcttta agggcgtgtc tcgtgaactg tatatcccga tgggtgccgc tctggccatt
     5941 tacgctcgcg agaacggcga tggtgtgatg ttcgaaccag aagaaatcta tgacgagctg
     6001 aatattggtt aaacatgttt aagggttccg gttccactag gtacaattcg atatcaagct
     6061 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     6121 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     6181 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     6241 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     6301 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     6361 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     6421 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     6481 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     6541 ggccctcaat ccagcggacc ttccttcccg cgcggcctgc tgccggctct gcggcctctt
     6601 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     6661 cgataccgtc gacctcgatc gagacctaga aaaacatgga gcaatcacaa gtagcaatac
     6721 agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt
     6781 tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
     6841 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     6901 tatccttgat ctgtggatct accacacaca aggctacttc cctgattggc agaactacac
     6961 accagggcca gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt
     7021 tgagcaagag aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt
     7081 gagcctgcat gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg
     7141 cctagcattt catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt
     7201 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     7261 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     7321 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa
     7381 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     7441 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     7501 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     7561 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     7621 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     7681 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     7741 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     7801 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     7861 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     7921 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     7981 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     8041 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     8101 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     8161 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     8221 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     8281 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     8341 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     8401 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     8461 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     8521 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     8581 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     8641 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     8701 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     8761 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     8821 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     8881 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     8941 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     9001 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     9061 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     9121 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     9181 c
//
