LOCUS       sequence-42927-e        6499 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Mid2(SS/TM), containing only the signal sequence and 
            transmembrane helix, fused to GFP and LOVpep..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6499)
  AUTHORS   Strickland D, Lin Y, Wagner E, Hope CM, Zayner J, Antoniou C, 
            Sosnick TR, Weiss EL, Glotzer M
  TITLE     TULIPs: tunable, light-controlled interacting protein tags for cell 
            biology.
  JOURNAL   Nat Methods. 2012 Mar 4. doi: 10.1038/nmeth.1904.
  PUBMED    22388287
REFERENCE   2  (bases 1 to 6499)
  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"; journalName: "Nat 
            Methods. 2012 Mar 4. doi: 10.1038/nmeth.1904."
COMMENT     Sequence Label: pDS257
FEATURES             Location/Qualifiers
     source          1..6499
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        22..463
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    22..139
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     434..457
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             792..1505
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(822..850)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1445..1465
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1984..2231
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2000..2018)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2256..2272)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2256..2272)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2269..2291)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2280..2296
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2304..2310,2311..2328,2329..2334))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2349..2370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2487..2504)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2658..3246)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2738..2757)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3417..4208,4209..4277))
                     /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     4040..4059
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4278..4382)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4450..4468
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4470..4874
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             4887..5981
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(6122..6144)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6244..6263
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6457..6479
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6471..6488
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6472..6488
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cgtttaacgg cgcgcctagt acggattaga agccgccgag cgggtgacag ccctccgaag
       61 gaagactctc ctccgtgcgt cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc
      121 ctcgcgccgc actgctccga acaataaaga ttctacaata ctagctttta tggttatgaa
      181 gaggaaaaat tggcagtaac ctggccccac aaaccttcaa atgaacgaat caaattaaca
      241 accataggat gataatgcga ttagtttttt agccttattt ctggggtaat taatcagcga
      301 agcgatgatt tttgatctat taacagatat ataaatgcaa aaactgcata accactttaa
      361 ctaatacttt caacattttc ggtttgtatt acttcttatt caaatgtaat aaaagtatca
      421 acaaaaaatt gttaatatac ctctatactt taacgtcaag gagaaaaacc ccggattctg
      481 aagcggccgc actagtggat cccccgggct gcaggaattc gatatcaagc ttatcgatac
      541 cgtcgacaat gttgtctttc acaaccaaga atagtttccg cttattactt ttaatactgt
      601 catgcatatc gacgatacgc gcacaatttt tcgtgcaatc atcatcttcg aattcgggtc
      661 tttctaaaaa aaatagaaac atcgtcattg gttgtgtggt tggtattggt gtaccactga
      721 ttctggtgat actggcttta atttacatgt tttgtatcca atcatcaagg acggacgcgt
      781 cgacctcgac aatgagtaaa ggagaagaac ttttcactgg agttgtccca attcttgttg
      841 aattagatgg tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg
      901 caacatacgg aaaacttacc cttaaattta tttgcactac tggaaaacta cctgttccat
      961 ggccaacact tgtcactact ttcacttatg gtgttcaatg cttttcaaga tacccagatc
     1021 atatgaaaca gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaaagaa
     1081 ctatattttt caaagatgac gggaactaca agacacgtgc tgaagtcaag tttgaaggtg
     1141 atacccttgt taatagaatc gagttaaaag gtattgattt taaagaagat ggaaacattc
     1201 ttggacacaa attggaatac aactataact cacacaatgt atacatcatg gcagacaaac
     1261 aaaagaatgg aatcaaagtt aacttcaaaa ttagacacaa cattgaagat ggaagcgttc
     1321 aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag
     1381 acaaccatta cctgtccaca caatctgccc tttcgaaaga tcccaacgaa aagagagacc
     1441 acatggtcct tcttgagttt gtaacagctg ctgggattac acatggcatg gatgaactat
     1501 acaaaggtgg atctggaggt tcaggtggaa gtcctaggtt ggctactaca cttgaacgta
     1561 ttgagaagaa ctttgtcatt actgacccaa gattgccaga taatcccatt atattcgcgt
     1621 ccgatagttt cttgcagttg acagaatata gccgtgaaga aattttggga agaaactgca
     1681 ggtttctaca aggtcctgaa actgatcgcg cgacagtgag aaaaattaga gatgccatag
     1741 ataaccaaac agaggtcact gttcagctga ttaattatac aaagagtggt aaaaagttct
     1801 ggaacctctt tcacttgcag cctatgcgag atcagaaggg agatgtccag tactttattg
     1861 gggttcagtt ggatggaact gagcatgtcc gagatgctgc cgagagagag gcagtcatgc
     1921 tgattaagaa aactgcagaa gagattgata aagcggtgga tacctgggtg taagcttctc
     1981 gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
     2041 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     2101 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     2161 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
     2221 ctttaatttg cggccgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     2281 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     2341 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     2401 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2461 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2521 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2581 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2641 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2701 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2761 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2821 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2881 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2941 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3001 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3061 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     3121 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3181 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3241 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3301 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3361 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3421 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3481 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3541 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3601 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3661 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3721 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3781 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3841 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3901 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3961 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4021 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4081 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4141 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4201 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4261 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4321 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4381 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     4441 acctataaaa ataggcgtat cacgaggcca actgtgggaa tactcaggta tcgtaagatg
     4501 caagagttcg aatctcttag caaccattat ttttttcctc aacataacga gaacacacag
     4561 gggcgctatc gcacagaatc aaattcgatg actggaaatt ttttgttaat ttcagaggtc
     4621 gcctgacgca tatacctttt tcaactgaaa aattgggaga aaaaggaaag gtgagaggcc
     4681 ggaaccggct tttcatatag aatagagaag cgttcatgac taaatgcttg catcacaata
     4741 cttgaagttg acaatattat ttaaggacct attgtttttt ccaataggtg gttagcaatc
     4801 gtcttacttt ctaacttttc ttacctttta catttcagca atatatatat atatttcaag
     4861 gatataccat tctaatgtct gcccctatgt ctgcccctaa gaagatcgtc gttttgccag
     4921 gtgaccacgt tggtcaagaa atcacagccg aagccattaa ggttcttaaa gctatttctg
     4981 atgttcgttc caatgtcaag ttcgatttcg aaaatcattt aattggtggt gctgctatcg
     5041 atgctacagg tgtcccactt ccagatgagg cgctggaagc ctccaagaag gttgatgccg
     5101 ttttgttagg tgctgtgggt ggtcctaaat ggggtacagg tagtgttaga cctgaacaag
     5161 gtttactaaa aatccgtaaa gaacttcaat tgtacgccaa cttaagacca tgtaactttg
     5221 catccgactc tcttttagac ttatctccaa tcaagccaca atttgctaaa ggtactgact
     5281 tcgttgttgt cagagaatta gtgggaggta tttactttgg taagagaaag gaagacgatg
     5341 gtgatggtgt cgcttgggat agtgaacaat acaccgttcc agaagtgcaa agaatcacaa
     5401 gaatggccgc tttcatggcc ctacaacatg agccaccatt gcctatttgg tccttggata
     5461 aagctaatgt tttggcctct tcaagattat ggagaaaaac tgtggaggaa accatcaaga
     5521 acgaatttcc tacattgaag gttcaacatc aattgattga ttctgccgcc atgatcctag
     5581 ttaagaaccc aacccaccta aatggtatta taatcaccag caacatgttt ggtgatatca
     5641 tctccgatga agcctccgtt atcccaggtt ccttgggttt gttgccatct gcgtccttgg
     5701 cctctttgcc agacaagaac accgcatttg gtttgtacga accatgccac ggttctgctc
     5761 cagatttgcc aaagaataag gttgacccta tcgccactat cttgtctgct gcaatgatgt
     5821 tgaaattgtc attgaacttg cctgaagaag gtaaggccat tgaagatgca gttaaaaagg
     5881 ttttggatgc aggtatcaga actggtgatt taggtggttc caacagtacc accgaagtcg
     5941 gtgatgctgt cgccgaagaa gttaagaaaa tccttgctta aaaagattct ctttttttat
     6001 gatatttgta cataaacttt ataaatgaaa ttcataatag aaacgacacg aaattacaaa
     6061 atggaatatg ttcatagggt aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     6121 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     6181 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     6241 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     6301 gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt
     6361 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     6421 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     6481 cggccagtga attcgagct
//
