LOCUS       sequence-173220-        8620 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ONM/ER split GFP11 reporter with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8620)
  AUTHORS   Smoyer CJ, Katta SS, Gardner JM, Stoltz L, McCroskey S, Bradford WD,
            McClain M, Smith SE, Slaughter BD, Unruh JR, Jaspersen SL
  TITLE     Analysis of membrane proteins localizing to the inner nuclear 
            envelope in living cells.
  JOURNAL   J Cell Biol. 2016 Nov 21;215(4):575-590. Epub 2016 Nov 9.
  PUBMED    27831485
REFERENCE   2  (bases 1 to 8620)
  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: "J Cell 
            Biol."; date: "2016-11-21"; volume: "215(4)"; pages: "575-590. Epub 
            2016 Nov 9"
COMMENT     Sequence Label: pSJ1568 (pRS315-NOP1pr-GFP11-mCherry-SCS2TM)
FEATURES             Location/Qualifiers
     source          1..8620
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1..17)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1..17)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14..36)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    25..41
                     /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(49..55,56..73,74..79))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    94..115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        181..556
                     /gene="S. cerevisiae ADE2"
                     /label=ADE2 promoter
     promoter        748..963
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             complement(1527..2621)
                     /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
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMSKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTAEVGDAVAEEVKKILA"
     promoter        complement(2622..3029)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(3815..3832)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4814..5402)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4894..4913)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5573..6364,6365..6433))
                     /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     6196..6215
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6434..6538)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6606..6624
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6662..6684)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6997..7019
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7011..7028
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7012..7028
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7059..7075
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7059..7075
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             7727..7774
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
ORIGIN
        1 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
       61 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
      121 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcaga tctgaattaa
      181 ttcttgaata atacataact tttcttaaaa gaatcaaaga cagataaaat ttaagagata
      241 ttaaatatta gtgagaagcc gagaattttg taacaccaac ataacactga catctttaac
      301 aacttttaat tatgatacat ttcttacgtc atgattgatt attacagcta tgctgacaaa
      361 tgactcttgt tgcatggcta cgaaccgggt aatactaagt gattgactct tgctgacctt
      421 ttattaagaa ctaaatggac aatattatgg agcatttcat gtataaattg gtgcgtaaaa
      481 tcgttggatc tctcttctaa gtacatccta ctataacaat caagaaaaac aagaaaatcg
      541 gacaaaacaa tcaagtatgg attctagaac agttggtata ttaggagggg gacaattggg
      601 acgtatgatt gttgaggcag caaacaggct caacattaag acggtaatac tagatgctga
      661 aaattctcct gccaaacaaa taagcaactc caatgaccac gttaatgcat cagagcagat
      721 tgtactgaga gtgcaccata ccaccttttc aattcatcat ttttttttta ttcttttttt
      781 tgatttcggt ttccttgaaa tttttttgat tcggtaatct ccgaacagaa ggaagaacga
      841 aggaaggagc acagacttag attggtatat atacgcatat gtagtgttga agaaacatga
      901 aattgcccag tattcttaac ccaactgcac agaacaaaaa cctgcaggaa acgaagataa
      961 atcatgtcga aagctacata taaggaacgt gctgctactc atcctagtcc tgttgctgcc
     1021 aaggcagatt gtactgagag tgcaccatat cgactacgtc gttaaggccg tttctgacag
     1081 agtaaaattc ttgagggaac tttcaccatt atgggaaatg gttcaagaag gtattgactt
     1141 aaactccatc aaatggtcag gtcattgagt gttttttatt tgttgtattt tttttttttt
     1201 agagaaaatc ctccaatata taaattagga atcatagttt catgattttc tgttacacct
     1261 aactttttgt gtggtgccct cctcctcgtc aatattaatg ttaaagtgca attctttttc
     1321 cttatcacgt tgagccatta gtatcaattt gcttacctgt attcctttac atcctccttt
     1381 ttctccttct tgataaatgt atgtagattg cgtatatagt ttcgtctacc ctatgaacat
     1441 attccatttt gtaatttcgt gtcgtttcta ttatgaattt catttataaa gtttatgtac
     1501 aaatatcata aaaaaagaga atctttttaa gcaaggattt tcttaacttc ttcggcgaca
     1561 gcatcaccga cttcggcggt actgttggaa ccacctaaat caccagttct gatacctgca
     1621 tccaaaacct ttttaactgc atcttcaatg gccttacctt cttcaggcaa gttcaatgac
     1681 aatttcgaca tcattgcagc agacaagata gtggcgatag ggttgacctt attctttggc
     1741 aaatctggag cagaaccgtg gcatggttcg tacaaaccaa atgcggtgtt cttgtctggc
     1801 aaagaggcca aggacgcaga tggcaacaaa cccaaggaac ctgggataac ggaggcttca
     1861 tcggagatga tatcaccaaa catgttgctg gtgattataa taccatttag gtgggttggg
     1921 ttcttaacta ggatcatggc ggcagaatca atcaattgat gttgaacctt caatgtaggg
     1981 aattcgttct tgatggtttc ctccacagtt tttctccata atcttgaaga ggccaaaaca
     2041 ttagctttat ccaaggacca aataggcaat ggtggctcat gttgtagggc catgaaagcg
     2101 gccattcttg tgattctttg cacttctgga acggtgtatt gttcactatc ccaagcgaca
     2161 ccatcaccat cgtcttcctt tctcttacca aagtaaatac ctcccactaa ttctctgaca
     2221 acaacgaagt cagtaccttt agcaaattgt ggcttgattg gagataagtc taaaagagag
     2281 tcggatgcaa agttacatgg tcttaagttg gcgtacaatt gaagttcttt acggattttt
     2341 agtaaacctt gttcaggtct aacactaccg gtaccccatt taggaccacc cacagcacct
     2401 aacaaaacgg catcagcctt cttggaggct tccagcgcct catctggaag tggaacacct
     2461 gtagcatcga tagcagcacc accaattaaa tgattttcga aatcgaactt gacattggaa
     2521 cgaacatcag aaatagcttt aagaacctta atggcttcgg ctgtgatttc ttgaccaacg
     2581 tggtcacctg gcaaaacgac gatcttctta ggggcagaca ttagaatggt atatccttga
     2641 aatatatata tatatattgc tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgat
     2701 tgctaaccac ctattggaaa aaacaatagg tccttaaata atattgtcaa cttcaagtat
     2761 tgtgatgcaa gcatttagtc atgaacgctt ctctattcta tatgaaaagc cggttccggc
     2821 gctctcacct ttcctttttc tcccaatttt tcagttgaaa aaggtatatg cgtcaggcga
     2881 cctctgaaat taacaaaaaa tttccagtca tcgaatttga ttctgtgcga tagcgcccct
     2941 gtgtgttctc gttatgttga ggaaaaaaat aatggttgct aagagattcg aactcttgca
     3001 tcttacgata cctgagtatt cccacagtta actgcggtca agatatttct tgaatcaggc
     3061 gccttagacc gctcggccaa acaaccaatt acttgttgag aaatagagta taattatcct
     3121 ataaatataa cgcttttgaa cacacatgaa cagggaagta caggacaatt gattttgaag
     3181 agaatgtgga ttttgatgta attgttggga ttccattttt aataaggcaa taatattagg
     3241 tatgtagata tactagaagt tctcctcgac cggtcgatat gcggtgtgaa ataccgcaca
     3301 gagggtgaac gttacagaaa agcaggctgg gaagcatatt tgagaagatg cggccagcaa
     3361 aactaaaaaa ctgtattata agtaaatgca tgtatactaa actcacaaat tagagcttca
     3421 atttaattat atcagttatt accctatgcg gtgtgaaata ccgcacagat gcgtaggaga
     3481 ttcaagttat acaacgaaaa tggaacagtt tttattaaag caagaagaag aagttcttgt
     3541 caaagcacaa aagttagaaa ctgtcggtta cgaagcttat ctagaaaaca agtaatatat
     3601 aagtttattg atatacttgt acagcaaata attataaaat gatataccta ttttttaggc
     3661 tttgttatga ttacatcaaa tgtggacttc atacatagaa atcaacgctt acaggtgtcc
     3721 ttttttaaga atttcataca taagatctgc tgcattaatg aatcggccaa cgcgcgggga
     3781 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3841 tcgttcggct gcggcgagcg gtatcagcat cgatgctaga gtcgaatgga aattacgtac
     3901 tttcaacgca agagcaagac aaaagaaagc ctaaaactgt cttttcgtgt cggtcgtcca
     3961 atatcatcgt aaacgtgtgt agagccttaa attctaattt ctaactttaa attttttttg
     4021 ctattataat actaatttct aactttcaac ctattttaca tcttcggaaa acaaaaacta
     4081 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
     4141 tttaaattat aattattttt atagcacgtg atgaaaagaa atatagtctt caaggttata
     4201 ttatacaggt ttatatatta ttttacttat ttttgttgtt ctttttcttg gagtatttag
     4261 cattaacaac ttcgacccat cgcgatgacg aaattgcacg tttaaaggag aagtcggaat
     4321 cgttgcaaga agaaataaat tagtttgacg ttttattcct tttcttctgc acagaagtgt
     4381 gtatatatat atttatatat agtcgatgtg caaaagccta cattattttt cttttgattt
     4441 ttattttttt gttcaagtag gttcatgccg acagtaaatc cttatgccct acctaaatat
     4501 aaacacccaa atatatatat atatatatat atatataaat aataaaaacg gaaaggaaat
     4561 agaataataa taataatact acacgtttac gtatgtaact cattctatta ttcagacttt
     4621 gatattaata gaattattac tactaggttt cctcacttca aaatctataa atataccgtc
     4681 atgctctaat cgatgctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4741 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4801 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4861 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4921 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4981 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5041 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5101 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5161 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5221 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5281 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5341 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5401 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5461 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5521 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5581 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5641 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5701 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5761 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5821 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5881 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5941 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6001 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6061 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6121 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6181 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6241 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6301 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6361 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6421 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6481 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6541 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     6601 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     6661 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     6721 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     6781 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     6841 gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt
     6901 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     6961 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     7021 cggccagtga attcgagctc caccgcggtg gcggccgctc tagaactagt ggatccattg
     7081 agtcatcagc ctcttcttga aaaaacgatt ctgaaataac cggaacttgt ttagattcct
     7141 ttttgtaggt taatcttatc ctgtacttgc tgatattgtg gttattagca gagatttttt
     7201 tcaaaacatc atctaaagta ggctttttgg ataagtcaat ttcagtgtcc cttaaccctt
     7261 tagagcggct ttttatggtg ataggcattt tcaaattaaa ttcaaaatat gtatctctct
     7321 caaatagctc aattggtttc tagatagcta aatagaatat aatcttactg tcctccgttc
     7381 tgtaaaattc acgctcttag tcccttttca taattcctta actttttgcg tacaaaatga
     7441 tatgtttatt atatttttct tttttttttt tcaaattttt tctttttctt gaaaaatttt
     7501 tcaaattgga aagctcatct ctcttgaatg tataatactt tcttcctcta actttcaaaa
     7561 agttttacat agccaagaag ttttccttac atcggtatac tactgttata taagttattc
     7621 ttcgagaaac aattagatat cattcatcgg ataaatctaa gttgcccatt gctttcaata
     7681 actccgatca aattaactca aatcaactaa aacagtaggg cccatgagag atcatatggt
     7741 tttgcatgaa tatgttaatg ctgctggtat tactggtggt ggttctggtg gtggttctct
     7801 cgagatggtt tctaaaggtg aagaagataa tatggctatt attaaagaat ttatgagatt
     7861 taaagttcat atggaaggtt ctgttaatgg tcatgaattt gaaattgaag gtgaaggtga
     7921 aggtagacca tatgaaggta ctcaaactgc taaattgaaa gttactaaag gtggtccatt
     7981 gccatttgct tgggatattt tgtctccaca atttatgtat ggttctaaag cttatgttaa
     8041 acatccagct gatattccag attatttgaa attgtctttt ccagaaggtt ttaaatggga
     8101 aagagttatg aattttgaag atggtggtgt tgttactgtt actcaagatt cttctttgca
     8161 agatggtgaa tttatttata aagttaaatt gagaggtact aattttccat ctgatggtcc
     8221 agttatgcaa aaaaaaacta tgggttggga agcttcttct gaaagaatgt atccagaaga
     8281 tggtgctttg aaaggtgaaa ttaaacaaag attgaaattg aaagatggtg gtcattatga
     8341 tgctgaagtt aaaactactt ataaagctaa aaaaccagtt caattgccag gtgcttataa
     8401 tgttaatatt aaattggata ttacttctca taatgaagat tatactattg ttgaacaata
     8461 tgaaagagct gaaggtagac attctactgg tggtatggat gaattgtata aaggtggtgg
     8521 tgctagctca tccagcatgg gtatattcat attggttgca ctccttatct tggttttagg
     8581 atggttctac agataagcat gcaagcttgg cgtaatcatg
//
