LOCUS       sequence-388470-        7740 bp    DNA     circular SYN 14-APR-2025
DEFINITION  pCYC-NLS-MCP-2xGFP on pRS305.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7740)
  AUTHORS   Li W, Maekiniemi A, Sato H, Osman C, Singer RH
  TITLE     An improved imaging system that corrects MS2-induced RNA 
            destabilization.
  JOURNAL   Nat Methods. 2022 Dec;19(12):1558-1562. doi: 
            10.1038/s41592-022-01658-1. Epub 2022 Nov 10.
  PUBMED    36357695
REFERENCE   2  (bases 1 to 7740)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-022-01658-1"; journalName: "Nat Methods"; date: 
            "2022-12"; volume: "19"; issue: "12"; pages: "1558-1562"
COMMENT     Sequence Label: pWL078 MCP-2xGFP
FEATURES             Location/Qualifiers
     source          1..7740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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     complement(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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)."
     primer_bind     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2913..2933
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2940..3650
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIKLKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             3660..4373
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             3660..4370
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     terminator      4404..4650
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4420..4438)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4662..4679)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4662..4678)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4671..4693)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4819..5274
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4906..4925)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5116..5137
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5575..5982
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5983..7077
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(7571..7590)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7690..7712
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctt gcatgcctgc agggagcgtt ggttggtgga tcaagcccac
     2281 gcgtaggcaa tcctcgagca gatccgccgg gcgtgtatat agcgtggatg gccaggcaac
     2341 tttagtgctg acacatacag gcatatatat atgtgtgcga cgacacatga tcatatggca
     2401 tgcatgtgct ctgtatgtat ataaaactct tgttttcttc ttttctctaa atattctttc
     2461 cttatacatt aggtcctttg tagcataaat tactatactt ctatagacac gcaaacacaa
     2521 atacacacac taaattaata ggatccatgc tagccgttaa aatggcttct aactttactc
     2581 agttcgttct cgtcgacaat ggcggaactg gcgacgtgac tgtcgcccca agcaacttcg
     2641 ctaacgggat cgctgaatgg atcagctcta actcgcgttc acaggcttac aaagtaacct
     2701 gtagcgttcg tcagagctct gcgcagaatc gcaaatacac catcaaagtc gaggtgccta
     2761 aaggcgcctg gcgttcgtac ttaaatatgg aactaaccat tccaattttc gccacgaatt
     2821 ccgactgcga gcttattgtt aaggcaatgc aaggtctcct aaaagatgga aacccgattc
     2881 cctcagcaat cgcagcaaac tccggcatct acccaaaaaa aaaaagaaaa gttaccggtt
     2941 ctaaaggtga agaattattc actggtgttg tcccaatttt ggttgaatta gatggtgatg
     3001 ttaatggtca caaattttct gtctccggtg aaggtgaagg tgatgctact tacggtaaat
     3061 tgaccttaaa atttatttgt actactggta aattgccagt tccatggcca accttagtca
     3121 ctactttaac ttatggtgtt caatgttttt ctagataccc agatcatatg aaacaacatg
     3181 actttttcaa gtctgccatg ccagaaggtt atgttcaaga aagaactatt tttttcaaag
     3241 atgacggtaa ctacaagacc agagctgaag tcaagtttga aggtgatacc ttagttaata
     3301 gaatcaaatt aaaaggtatt gattttaaag aagatggtaa cattttaggt cacaaattgg
     3361 aatacaacta taactctcac aatgtttaca tcatggctga caaacaaaag aatggtatca
     3421 aagttaactt caaaattaga cacaacattg aagatggttc tgttcaatta gctgaccatt
     3481 atcaacaaaa tactccaatt ggtgatggtc cagtcttgtt accagacaac cattacttat
     3541 ccactcaatc tgccttatcc aaagatccaa acgaaaagag agaccacatg gtcttgttag
     3601 aatttgttac tgctgctggt attacccatg gtatggatga attgtacaaa tgtttaaact
     3661 ctaaaggtga agaattattc actggtgttg tcccaatttt ggttgaatta gatggtgatg
     3721 ttaatggtca caaattttct gtctccggtg aaggtgaagg tgatgctact tacggtaaat
     3781 tgaccttaaa atttatttgt actactggta aattgccagt tccatggcca accttagtca
     3841 ctactttaac ttatggtgtt caatgttttt ctagataccc agatcatatg aaacaacatg
     3901 actttttcaa gtctgccatg ccagaaggtt atgttcaaga aagaactatt tttttcaaag
     3961 atgacggtaa ctacaagacc agagctgaag tcaagtttga aggtgatacc ttagttaata
     4021 gaatcgaatt aaaaggtatt gattttaaag aagatggtaa cattttaggt cacaaattgg
     4081 aatacaacta taactctcac aatgtttaca tcatggctga caaacaaaag aatggtatca
     4141 aagttaactt caaaattaga cacaacattg aagatggttc tgttcaatta gctgaccatt
     4201 atcaacaaaa tactccaatt ggtgatggtc cagtcttgtt accagacaac cattacttat
     4261 ccactcaatc tgccttatcc aaagatccaa acgaaaagag agaccacatg gtcttgttag
     4321 aatttgttac tgctgctggt attacccatg gtatggatga attgtacaaa taagtttaaa
     4381 cccgctgatc ctagagggcc gcatcatgta attagttatg tcacgcttac attcacgccc
     4441 tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc
     4501 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct
     4561 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa
     4621 ggttttggga cgctcgaagg ctttaatttg agctcgaatt cactggccgt cgttttacaa
     4681 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     4741 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     4801 agcctgaatg gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4861 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4921 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4981 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5041 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5101 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5161 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5221 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat
     5281 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatcga ccggtcgagg
     5341 agaacttcta gtatatctac atacctaata ttattgcctt attaaaaatg gaatcccaac
     5401 aattacatca aaatccacat tctcttcaaa atcaattgtc ctgtacttcc ttgttcatgt
     5461 gtgttcaaaa acgttatatt tataggataa ttatactcta tttctcaaca agtaattggt
     5521 tgtttggccg agcggtctaa ggcgcctgat tcaagaaata tcttgaccgc agttaactgt
     5581 gggaatactc aggtatcgta agatgcaaga gttcgaatct cttagcaacc attatttttt
     5641 tcctcaacat aacgagaaca cacaggggcg ctatcgcaca gaatcaaatt cgatgactgg
     5701 aaattttttg ttaatttcag aggtcgcctg acgcatatac ctttttcaac tgaaaaattg
     5761 ggagaaaaag gaaaggtgag agcgccggaa ccggcttttc atatagaata gagaagcgtt
     5821 catgactaaa tgcttgcatc acaatacttg aagttgacaa tattatttaa ggacctattg
     5881 ttttttccaa taggtggtta gcaatcgtct tactttctaa cttttcttac cttttacatt
     5941 tcagcaatat atatatatat atttcaagga tataccattc taatgtctgc ccctaagaag
     6001 atcgtcgttt tgccaggtga ccacgttggt caagaaatca cagccgaagc cattaaggtt
     6061 cttaaagcta tttctgatgt tcgttccaat gtcaagttcg atttcgaaaa tcatttaatt
     6121 ggtggtgctg ctatcgatgc tacaggtgtt ccacttccag atgaggcgct ggaagcctcc
     6181 aagaaggctg atgccgtttt gttaggtgct gtgggtggtc ctaaatgggg taccggtagt
     6241 gttagacctg aacaaggttt actaaaaatc cgtaaagaac ttcaattgta cgccaactta
     6301 agaccatgta actttgcatc cgactctctt ttagacttat ctccaatcaa gccacaattt
     6361 gctaaaggta ctgacttcgt tgttgtcaga gaattagtgg gaggtattta ctttggtaag
     6421 agaaaggaag acgatggtga tggtgtcgct tgggatagtg aacaatacac cgttccagaa
     6481 gtgcaaagaa tcacaagaat ggccgctttc atggccctac aacatgagcc accattgcct
     6541 atttggtcct tggataaagc taatgttttg gcctcttcaa gattatggag aaaaactgtg
     6601 gaggaaacca tcaagaacga attccctaca ttgaaggttc aacatcaatt gattgattct
     6661 gccgccatga tcctagttaa gaacccaacc cacctaaatg gtattataat caccagcaac
     6721 atgtttggtg atatcatctc cgatgaagcc tccgttatcc caggttcctt gggtttgttg
     6781 ccatctgcgt ccttggcctc tttgccagac aagaacaccg catttggttt gtacgaacca
     6841 tgccacggtt ctgctccaga tttgccaaag aataaggtca accctatcgc cactatcttg
     6901 tctgctgcaa tgatgttgaa attgtcattg aacttgcctg aagaaggtaa ggccattgaa
     6961 gatgcagtta aaaaggtttt ggatgcaggt atcagaactg gtgatttagg tggttccaac
     7021 agtaccaccg aagtcggtga tgctgtcgcc gaagaagtta agaaaatcct tgcttaaaaa
     7081 gattctcttt ttttatgata tttgtacata aactttataa atgaaattca taatagaaac
     7141 gacacgaaat tacaaaatgg aatatgttca tagggtagac gaaactatat acgcaatcta
     7201 catacattta tcaagaagga gaaaaaggag gatgtaaagg aatacaggta agcaaattga
     7261 tactaatggc tcaacgtgat aaggaaaaag aattgcactt taacattaat attgacaagg
     7321 aggagggcac cacacaaaaa gttaggtgta acagaaaatc atgaaactat gattcctaat
     7381 ttatatattg gaggattttc tctaaaaaaa aaaaaataca acaaataaaa aacactcaat
     7441 gacctgacca tttgatggag tttaagtcaa taccttcttg aaccatttcc cataatggtg
     7501 aaagttccct caagaatttt actctgtcag aaacggcctt aacgacgtag tcgatatggt
     7561 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     7621 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     7681 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
//
