LOCUS       sequence-98853-e        9110 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of ITSN DH/PH-tgRFPt-Nano.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9110)
  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 9110)
  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: hITSN1(1159-1509)-tgRFPt-SSPB WT
FEATURES             Location/Qualifiers
     source          1..9110
                     /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..2931
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2850..2870
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     2948..2964
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3006..3039)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        3155..3458
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3459..3662
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             4802..5530
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     CDS             5606..5935
                     /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    5997..6587
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6050..6070)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6468..6479)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6590..6606)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             7116..7296
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7358..7946)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7438..7457)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8117..8908,8909..8977))
                     /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     8740..8759
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8978..9082)
                     /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 ccgacgccgc catctctagg
     2641 cccgcgccgg ccccctcgca cagacttgtg ggagaagctc ggctactccc ctgccccggt
     2701 taatttgcat ataatatttc ctagtaacta tagaggctta atgtgcgata aaagacagat
     2761 aatctgttct ttttaatact agctacattt tacatgatag gcttggattt ctataagaga
     2821 tacaaatact aaattattat tttaaaaaac agcacaaaag gaaactcacc ctaactgtaa
     2881 agtaattgtg tgttttgaga ctataaatat cccttggaga aaagccttgt taacgcgcgg
     2941 tgaccctcga ggtcgacggt atcgataagc tcgcttcacg agattccagc aggtcgaggg
     3001 acctaataac ttcgtatagc atacattata cgaagttata ttaagggttc caagcttaag
     3061 cggccgcgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg
     3121 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     3181 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     3241 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     3301 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     3361 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     3421 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     3481 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     3541 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     3601 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     3661 ctggtttagt gaaccgtcag atccgctaga attctgcagt cgacggtacc gcgggcccgc
     3721 caccatgttg accccaactg aaagaaagcg acaaggatac atccacgagc tcattgtcac
     3781 cgaggagaac tatgtgaatg acctgcagct ggtcacagag atttttcaaa aacccctgat
     3841 ggagtctgag ctgctgacag aaaaagaggt tgctatgatt tttgtgaact ggaaggagct
     3901 gattatgtgt aatatcaaac tactaaaagc gctgagagtc cgcaagaaga tgtccgggga
     3961 gaagatgcct gtgaagatga ttggagacat cctgagcgca cagctgccgc acatgcagcc
     4021 ctacatccgc ttctgcagcc gccagctcaa cggggctgcc ctgatccagc agaagacgga
     4081 cgaggcccca gacttcaagg agttcgtcaa aagattggca atggatcctc ggtgtaaagg
     4141 gatgccactc tctagtttta tactgaagcc tatgcaacgg gtaacaagat acccactgat
     4201 cattaaaaat atcctggaaa acacccctga aaaccacccg gaccacagcc acttgaagca
     4261 cgccctggag aaggcggaag agctctgttc ccaggtgaac gaaggggtgc gggagaagga
     4321 gaactctgac cggctggagt ggatccaggc ccacgtgcag tgtgaaggcc tgtctgagca
     4381 acttgtgttc aattcagtga ccaattgctt ggggccgcgc aaatttctgc acagtgggaa
     4441 gctctacaag gccaagagca acaaggagct gtatggcttc cttttcaacg acttcctcct
     4501 gctgactcag atcacgaagc ctttggggtc ttctggcacc gacaaagtct tcagccccaa
     4561 atcaaacctg cagtataaaa tgtataaaac acctattttc ctaaatgagg ttctagtaaa
     4621 attacccacc gacccttctg gagacgagcc catcttccac atctcccaca ttgaccgcgt
     4681 ctatactctc cgagcagaaa gcataaatga aaggactgcc tgggtgcaga aaatcaaagc
     4741 tgcttctgaa ctctacatag agactgagaa aaagaaggaa ttcggtagtg gaagtggttc
     4801 cgtgtctaag ggcgaagagc tgattaagga gaacatgcac atgaagctgt acatggaggg
     4861 caccgtgaac aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
     4921 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     4981 cctggctacc agcttcatgt acggcagcag aaccttcatc aaccacaccc agggcatccc
     5041 cgacttcttt aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     5101 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     5161 caacgtcaag atcagagggg tgaacttccc atccaacggc cctgtgatgc agaagaaaac
     5221 actcggctgg gaggccaaca ccgagatgct gtaccccgct gacggcggcc tggaaggcag
     5281 aaccgacatg gccctgaagc tcgtgggcgg gggccacctg atctgcaact tcaagaccac
     5341 atacagatcc aagaaacccg ctaagaacct caagatgccc ggcgtctact atgtggacca
     5401 cagactggaa agaatcaagg aggccgacaa agagacctac gtcgagcagc acgaggtggc
     5461 tgtggccaga tactgcgacc tccctagcaa actggggcac aaacttaatg gcatggacga
     5521 gctgtacaag tccggactca gatctcgagc tcaagcttcg aacgaattcg gcattgatct
     5581 gagcggcctg accctgcagg aattcagctc cccgaaacgc cctaagctgc tgcgtgaata
     5641 ttacgattgg ctggttgata acagctttac cccatatctg gtggtggatg ccacatacct
     5701 gggcgtgaac gtgcccgtgg agtatgtgaa agacggtcag atcgtgctga atctgtctgc
     5761 aagtgcgacc ggcaacctgc aactgacaaa tgattttatc cagttcaacg cccgctttaa
     5821 gggcgtgtct cgtgaactgt atatcccgat gggtgccgct ctggccattt acgctcgcga
     5881 gaacggcgat ggtgtgatgt tcgaaccaga agaaatctat gacgagctga atattggtta
     5941 aacatgttta agggttccgg ttccactagg tacaattcga tatcaagctt atcgataatc
     6001 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     6061 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     6121 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     6181 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     6241 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     6301 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     6361 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     6421 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     6481 cagcggacct tccttcccgc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     6541 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg
     6601 acctcgatcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca
     6661 atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac
     6721 ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     6781 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc
     6841 tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag
     6901 ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga
     6961 aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg
     7021 ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc
     7081 atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct
     7141 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     7201 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     7261 tcagaccctt ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa
     7321 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     7381 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     7441 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     7501 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     7561 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     7621 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     7681 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     7741 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     7801 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     7861 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     7921 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     7981 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     8041 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     8101 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     8161 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     8221 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     8281 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     8341 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     8401 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     8461 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     8521 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     8581 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     8641 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     8701 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     8761 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     8821 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     8881 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     8941 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     9001 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     9061 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
//
