LOCUS       sequence-271004-        4849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a tandem dimer of WT SspB (nano), targeted 
            to membrane by Lyn myristoylation sequence, with triple-HA epitope 
            tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4849)
  AUTHORS   Hope JM, Liu A, Calvin GJ, Cui B
  TITLE     Construction of Light-Activated Neurotrophin Receptors Using the 
            Improved Light-Induced Dimerizer (iLID).
  JOURNAL   J Mol Biol. 2020 Jun 12;432(13):3739-3748. doi: 
            10.1016/j.jmb.2020.04.018. Epub 2020 Apr 23.
  PUBMED    32335036
REFERENCE   2  (bases 1 to 4849)
  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.jmb.2020.04.018"; journalName: "J Mol Biol"; date: 
            "2020-06-12- 12"; volume: "432"; issue: "13"; pages: "3739-3748"
COMMENT     Sequence Label: Lyn-tdnano-HA
FEATURES             Location/Qualifiers
     source          1..4849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      598..607
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             679..1008
                     /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"
     CDS             1066..1395
                     /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"
     CDS             1426..1452
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1456..1482
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1486..1512
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1637..1758
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1674..1693)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1728..1747
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1765..2220)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1902..1923)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2114..2133
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2247..2351
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2353..2710
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2353..2544
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2376..2396)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2561..2696
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2745..3539
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2745..3539
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2799..2818)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3409..3428
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3727..3746)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3771..3818
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4147..4735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4636..4655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcggcc
      601 accatgggat gtattaaatc aaaaaggaaa gacaatctca atgacggtgg aagcggagga
      661 agcggtggtt cagaattcag ctccccgaaa cgccctaagc tgctgcgtga atattacgat
      721 tggctggttg ataacagctt taccccatat ctggtggtgg atgccacata cctgggcgtg
      781 aacgtgcccg tggagtatgt gaaagacggt cagatcgtgc tgaatctgtc tgcaagtgcg
      841 accggcaacc tgcaactgac aaatgatttt atccagttca acgcccgctt taagggcgtg
      901 tctcgtgaac tgtatatccc gatgggtgcc gctctggcca tttacgctcg cgagaacggc
      961 gatggtgtga tgttcgaacc agaagaaatc tatgacgagc tgaatattgg aggcggctcc
     1021 ggaggcagcg gcggttctgg aggttccggt ggcggctccg aattcagctc cccgaaacgc
     1081 cctaagctgc tgcgtgaata ttacgattgg ctggttgata acagctttac cccatatctg
     1141 gtggtggatg ccacatacct gggcgtgaac gtgcccgtgg agtatgtgaa agacggtcag
     1201 atcgtgctga atctgtctgc aagtgcgacc ggcaacctgc aactgacaaa tgattttatc
     1261 cagttcaacg cccgctttaa gggcgtgtct cgtgaactgt atatcccgat gggtgccgct
     1321 ctggccattt acgctcgcga gaacggcgat ggtgtgatgt tcgaaccaga agaaatctat
     1381 gacgagctga atattggagg aggaagtggt ggaagtggtg gtagttaccc gtatgatgtt
     1441 ccggattacg ctggctatcc ctacgacgtg cccgactatg ccgggtaccc ctatgacgtc
     1501 ccagactacg catgagcggc cgcgactcta gatcataatc agccatacca catttgtaga
     1561 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     1621 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     1681 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     1741 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     1801 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     1861 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     1921 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     1981 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     2041 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     2101 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     2161 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     2221 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2281 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2341 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     2401 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2461 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2521 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     2581 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2641 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2701 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     2761 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     2821 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     2881 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     2941 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     3001 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     3061 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     3121 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     3181 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     3241 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     3301 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     3361 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     3421 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     3481 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     3541 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     3601 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     3661 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     3721 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     3781 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     3841 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     3901 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     3961 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     4021 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4081 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4141 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4201 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4261 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4321 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4381 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4441 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4501 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4561 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4621 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4681 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4741 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4801 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
