LOCUS       sequence-178564-        7521 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  ish1-mCherry insertion cassette with NAT resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7521)
  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 7521)
  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: pMGF170
FEATURES             Location/Qualifiers
     source          1..7521
                     /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..3434
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        2315..2658
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2659..3231
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      3237..3434
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(4058..4080)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4225..4243)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4311..4415
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4416..4484,4485..5276)
                     /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(4634..4653)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5447..6035
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5936..5955
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6189..6206
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6279..6356
                     /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             6357..7439
                     /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(6376..6395)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    7452..7473
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7488..7493,7494..7511,7512..7518)
                     /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 aaacaaccat gggtaccact cttgacgaca cggcttaccg gtaccgcacc
     2701 agtgtcccgg gggacgccga ggccatcgag gcactggatg ggtccttcac caccgacacc
     2761 gtcttccgcg tcaccgccac cggggacggc ttcaccctgc gggaggtgcc ggtggacccg
     2821 cccctgacca aggtgttccc cgacgacgaa tcggacgacg aatcggacga cggggaggac
     2881 ggcgacccgg actcccggac gttcgtcgcg tacggggacg acggcgacct ggcgggcttc
     2941 gtggtcgtct cgtactccgg ctggaaccgc cggctgaccg tcgaggacat cgaggtcgcc
     3001 ccggagcacc gggggcacgg ggtcgggcgc gcgttgatgg ggctcgcgac ggagttcgcc
     3061 cgcgagcggg gcgccgggca cctctggctg gaggtcacca acgtcaacgc accggcgatc
     3121 cacgcgtacc ggcggatggg gttcaccctc tgcggcctgg acaccgccct gtacgacggc
     3181 accgcctcgg acggcgagca ggcgctctac atgagcatgc cctgccccta atcagtactg
     3241 acaataaaaa gattcttgtt ttcaagaact tgtcatttgt atagtttttt tatattgtag
     3301 ttgttctatt ttaatcaaat gttagcgtga tttatatttt ttttcgcctc gacatcatct
     3361 gcccagatgc gaagttaagt gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc
     3421 tggtcgctat actgctgtcg attcgatact aacgccgcca tccataaatt gttcatgaac
     3481 gttcttaatg tctatttata ttatttaaaa agaaggagat tacgtctaaa acgagttctc
     3541 gtttaatata tgacttttaa tgaaatatct atgttcatat tcgctactta accttccttg
     3601 gcaagtaatc catcctattg ttaacattaa tataatccat caagatactt ttttattaat
     3661 ttaagttttt cagaattaga tgttggtgca cctcctccaa atctagagct gtggaaagaa
     3721 ttggtaaaat cttatcgcga tgttcgtgat tctctaaaaa ttgcagtaaa acatttctag
     3781 catattcttt gttaatcaat tctttagaaa tattgcttat aacaggtggc tcgaatgaac
     3841 ctgctgaatc acttgaagct ttgggcaata tttcactgct cataacactg gacagctcgc
     3901 tctcgtgaga tttgacactt tccttttgaa ccttttgctt ttcaatgagt tcttttattt
     3961 catcccttga ctccactaac tcgtttccaa cactcgagcg gccgcatcgt gactgactga
     4021 cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     4081 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     4141 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     4201 tgtataattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     4261 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     4321 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     4381 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     4441 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     4501 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     4561 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     4621 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     4681 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     4741 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     4801 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     4861 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     4921 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     4981 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     5041 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     5101 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     5161 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     5221 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     5281 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     5341 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     5401 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5461 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5521 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5581 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5641 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5701 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5761 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5821 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5881 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5941 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6001 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6061 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     6121 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     6181 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     6241 ttacgcatct gtgcggtatt tcacaccgca taaattccga caccatcgaa tggtgcaaaa
     6301 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     6361 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     6421 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     6481 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     6541 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     6601 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     6661 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     6721 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     6781 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     6841 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     6901 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     6961 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     7021 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     7081 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     7141 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     7201 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     7261 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     7321 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     7381 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     7441 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     7501 cttccggctc gtatgttgtg t
//
