LOCUS       sequence-254723-        8094 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of tgRFPt-Nano.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8094)
  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 8094)
  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: tgRFPt-SSPB WT
FEATURES             Location/Qualifiers
     source          1..8094
                     /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      3699..3708
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3705..4436
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     CDS             4512..4841
                     /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"
     protein_bind    complement(4896..4929)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4985..5571
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5038..5058)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5456..5467)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5574..5590)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             6100..6280
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6342..6930)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6422..6441)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7101..7892,7893..7961))
                     /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     7724..7743
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7962..8066)
                     /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 gaccggtcgc caccatggtg tctaagggcg
     3721 aagagctgat taaggagaac atgcacatga agctgtacat ggagggcacc gtgaacaacc
     3781 accacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga
     3841 gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctaccagct
     3901 tcatgtacgg cagcagaacc ttcatcaacc acacccaggg catccccgac ttctttaagc
     3961 agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc
     4021 tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca
     4081 gaggggtgaa cttcccatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg
     4141 ccaacaccga gatgctgtac cccgctgacg gcggcctgga aggcagaacc gacatggccc
     4201 tgaagctcgt gggcgggggc cacctgatct gcaacttcaa gaccacatac agatccaaga
     4261 aacccgctaa gaacctcaag atgcccggcg tctactatgt ggaccacaga ctggaaagaa
     4321 tcaaggaggc cgacaaagag acctacgtcg agcagcacga ggtggctgtg gccagatact
     4381 gcgacctccc tagcaaactg gggcacaaac ttaatggcat ggacgagctg tacaagtccg
     4441 gactcagatc tcgagctcaa gcttcgaacg aattcggcat tgatctgagc ggcctgaccc
     4501 tgcaggaatt cagctccccg aaacgcccta agctgctgcg tgaatattac gattggctgg
     4561 ttgataacag ctttacccca tatctggtgg tggatgccac atacctgggc gtgaacgtgc
     4621 ccgtggagta tgtgaaagac ggtcagatcg tgctgaatct gtctgcaagt gcgaccggca
     4681 acctgcaact gacaaatgat tttatccagt tcaacgcccg ctttaagggc gtgtctcgtg
     4741 aactgtatat cccgatgggt gccgctctgg ccatttacgc tcgcgagaac ggcgatggtg
     4801 tgatgttcga accagaagaa atctatgacg agctgaatat tggttaagga tccaccggat
     4861 caagataacc tgcaggcaat tcgtcgaggg acctaataac ttcgtatagc atacattata
     4921 cgaagttata catgtttaag ggttccggtt ccactaggta caattcgata tcaagcttat
     4981 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     5041 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     5101 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     5161 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     5221 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     5281 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     5341 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     5401 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     5461 cctcaatcca gcggaccttc cttcccggcc tgctgccggc tctgcggcct cttccgcgtc
     5521 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     5581 gtcgacctcg atcgagacct agaaaaacat ggagcaatca caagtagcaa tacagcagct
     5641 accaatgctg attgtgcctg gctagaagca caagaggagg aggaggtggg ttttccagtc
     5701 acacctcagg tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     5761 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatatcctt
     5821 gatctgtgga tctaccacac acaaggctac ttccctgatt ggcagaacta cacaccaggg
     5881 ccagggatca gatatccact gacctttgga tggtgctaca agctagtacc agttgagcaa
     5941 gagaaggtag aagaagccaa tgaaggagag aacacccgct tgttacaccc tgtgagcctg
     6001 catgggatgg atgacccgga gagagaagta ttagagtgga ggtttgacag ccgcctagca
     6061 tttcatcaca tggcccgaga gctgcatccg gactgtactg ggtctctctg gttagaccag
     6121 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     6181 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     6241 tccctcagac ccttttagtc agtgtggaaa atctctagca gcatgtgagc aaaaggccag
     6301 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     6361 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6421 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6481 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6541 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6601 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6661 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6721 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6781 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6841 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6901 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6961 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     7021 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     7081 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     7141 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     7201 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     7261 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7321 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     7381 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7441 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7501 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7561 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7621 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7681 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7741 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7801 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7861 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7921 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7981 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     8041 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgac
//
