LOCUS       sequence-178562-        7757 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  ish1 mCherry insertion cassette with G418 resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7757)
  AUTHORS   Gu M, LaJoie D, Chen OS, von Appen A, Ladinsky MS, Redd MJ, Nikolova
            L, Bjorkman PJ, Sundquist WI, Ullman KS, Frost A
  TITLE     LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in 
            fission yeast and human cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Mar 14;114(11):E2166-E2175. doi: 
            10.1073/pnas.1613916114. Epub 2017 Feb 27.
  PUBMED    28242692
REFERENCE   2  (bases 1 to 7757)
  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.1613916114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-03-14- 14"; volume: "114"; issue: "11"; pages: 
            "E2166-E2175"
COMMENT     Sequence Label: pMGF169
FEATURES             Location/Qualifiers
     source          1..7757
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    5..21
                     /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     10..32
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             44..697
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     655..677
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             704..727
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1322..2032
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1467..1485)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1735..1754
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      2053..2240
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2108..2132)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            2315..3670
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2315..2657
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2658..3467
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2725..2744)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3473..3670
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(4294..4316)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4461..4479)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4547..4651
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4652..4720,4721..5512)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4870..4889)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5683..6271
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6172..6191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6425..6442
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6515..6592
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             6593..7675
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(6612..6631)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    7688..7709
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7724..7729,7730..7747,7748..7754)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 ggaattgtga gcggataaca atttcacaca ggaaacagta ttcatgtccc ctatactagg
       61 ttattggaaa attaagggcc ttgtgcaacc cactcgactt cttttggaat atcttgaaga
      121 aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt
      181 tgaattgggt ttggagtttc ccaatcttcc ttattatatt gatggtgatg ttaaattaac
      241 acagtctatg gccatcatac gttatatagc tgacaagcac aacatgttgg gtggttgtcc
      301 aaaagagcgt gcagagattt caatgcttga aggagcggtt ttggatatta gatacggtgt
      361 ttcgagaatt gcatatagta aagactttga aactctcaaa gttgattttc ttagcaagct
      421 acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt taaatggtga
      481 tcatgtaacc catcctgact tcatgttgta tgacgctctt gatgttgttt tatacatgga
      541 cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat
      601 cccacaaatt gataagtact tgaaatccag caagtatata gcatggcctt tgcagggctg
      661 gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg gatctggaag ttctgttcca
      721 ggggcccctg gctgttccta attgggctgc ttattttgat actttgagct ccaaagagct
      781 ttctgcctgg attaaggaat acaaaaaaca ttaccatgga aaacttcacc catccaaaga
      841 tcgtgaacat cttttccaaa atgcctgtaa tatttatcgc gctattgctg aaaaggctga
      901 caaatctgtt cttcaatcga ttcaatcaaa gtttcctaag gcaactgttc ccggttatga
      961 ctcttggtct aatgaagatc taaagtctgc actcaaggaa tatggtgaca gtattggtaa
     1021 ggtgtttaat cgtaaagatg ctattgaacg cttaaagcgt catgatattt tattctatgg
     1081 tcctgttgtt gccgacaagg ctaaatcagg agtctctggc ttccttcgac gtgttgcttc
     1141 ttttatggga tttggcactc gcgacgtagc tgaaattaag ctaggagaaa acattcaaaa
     1201 ggtaaaagat actgcctcta aagcctcatc tgctgtatct tcagcaggcg actatgtcga
     1261 aactaatgta aagaaagccc aaagggtttt gggaggaacg ggcggtagtg gaggcactgg
     1321 tatggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
     1381 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     1441 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
     1501 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
     1561 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     1621 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     1681 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     1741 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
     1801 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
     1861 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
     1921 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     1981 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt gaggcgcgcc
     2041 acttctaaat aagcgaattt cttatgattt atgattttta ttattaaata agttataaaa
     2101 aaaataagtg tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa attcttattc
     2161 ttgagtaact ctttcctgta ggtcaggttg ctttctcagg tatagtatga ggtcgctctt
     2221 attgaccaca cctctaccgg cagatccgct agggataaca gggtaatata gatctgttta
     2281 gcttgcctcg tccccgccgg gtcacccggc cagcgacatg gaggcccaga ataccctcct
     2341 tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt acatttagcc
     2401 catacatccc catgtataat catttgcatc catacatttt gatggccgca cggcgcgaag
     2461 caaaaattac ggctcctcgc tgcagacctg cgagcaggga aacgctcccc tcacagacgc
     2521 gttgaattgt ccccacgccg cgcccctgta gagaaatata aaaggttagg atttgccact
     2581 gaggttcttc tttcatatac ttccttttaa aatcttgcta ggatacagtt ctcacatcac
     2641 atccgaacat aaacaacatg ggtaaggaaa agactcacgt ttcgaggccg cgattaaatt
     2701 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     2761 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     2821 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     2881 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     2941 tcaccactgc gatccccggc aaaacagcat tccaggtatt agaagaatat cctgattcag
     3001 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     3061 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     3121 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     3181 aagtctggaa agaaatgcat aagcttttgc cattctcacc ggattcagtc gtcactcatg
     3241 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     3301 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     3361 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     3421 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gtactgacaa
     3481 taaaaagatt cttgttttca agaacttgtc atttgtatag tttttttata ttgtagttgt
     3541 tctattttaa tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc
     3601 agatgcgaag ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt
     3661 cgctatactg ctgtcgattc gatactaacg ccgccatcca taaattgttc atgaacgttc
     3721 ttaatgtcta tttatattat ttaaaaagaa ggagattacg tctaaaacga gttctcgttt
     3781 aatatatgac ttttaatgaa atatctatgt tcatattcgc tacttaacct tccttggcaa
     3841 gtaatccatc ctattgttaa cattaatata atccatcaag atactttttt attaatttaa
     3901 gtttttcaga attagatgtt ggtgcacctc ctccaaatct agagctgtgg aaagaattgg
     3961 taaaatctta tcgcgatgtt cgtgattctc taaaaattgc agtaaaacat ttctagcata
     4021 ttctttgtta atcaattctt tagaaatatt gcttataaca ggtggctcga atgaacctgc
     4081 tgaatcactt gaagctttgg gcaatatttc actgctcata acactggaca gctcgctctc
     4141 gtgagatttg acactttcct tttgaacctt ttgcttttca atgagttctt ttatttcatc
     4201 ccttgactcc actaactcgt ttccaacact cgagcggccg catcgtgact gactgacgat
     4261 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     4321 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     4381 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     4441 taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4501 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4561 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4621 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4681 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4741 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4801 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4861 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     4921 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4981 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5041 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     5101 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5161 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     5221 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     5281 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5341 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5401 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5461 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5521 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5581 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5641 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5701 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5761 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5821 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5881 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5941 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6001 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6061 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6121 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6181 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6241 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6301 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6361 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     6421 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     6481 gcatctgtgc ggtatttcac accgcataaa ttccgacacc atcgaatggt gcaaaacctt
     6541 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     6601 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     6661 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     6721 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     6781 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     6841 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     6901 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     6961 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     7021 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     7081 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     7141 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     7201 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     7261 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     7321 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     7381 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     7441 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     7501 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     7561 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     7621 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     7681 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     7741 cggctcgtat gttgtgt
//
