LOCUS       sequence-178532-        6982 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  S. pombe lem2 knockout cassette with hygro resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6982)
  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 6982)
  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: pMGF130
FEATURES             Location/Qualifiers
     source          1..6982
                     /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"
     gene            1334..2909
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1334..1677
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1678..2706
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1678..2706
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      2712..2909
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3519..3541)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3686..3704)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3772..3876
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3877..3945,3946..4737)
                     /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(4095..4114)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4908..5496
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5397..5416
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5650..5667
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5740..5817
                     /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             5818..6900
                     /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(5837..5856)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    6913..6934
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6949..6954,6955..6972,6973..6979)
                     /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 ctcgacatcg ctggaagcaa ccattgctcc gcccggttta gaattaattt
      781 tcttcttatt acgttgaaat ctacgtccaa tgtctttttg cattttttac tatagtcaaa
      841 attcgagtaa tccctaatga tcatggattc tgtattctta gtgattgatg ttggttaagg
      901 ataacttcgg gggcaacact cttacccaat atacgttatt tgggctgagg tgacacaaag
      961 caatgagctg cgctatgcct ccttatagag ctttcttccc ttaaccatta tttgccaccc
     1021 actttaactg gcaaaaagaa agaaggaaaa caaagattcg gcacaaagat agagaaataa
     1081 gaaattgctt taccttcgtt tagtgaagtc ttttattaaa tttgaaaagg tgtgtttgaa
     1141 cagtggaatt caagcaattt cctaagtata ttatagctca ttgctgtaat attataaatt
     1201 tgtatgggat tgcaattacg ttcacgaatc agcaacttat gccaggtttc ctttttaatg
     1261 tagataagga ttcatgtaat taactagctt atatcagctt tataccattt ttgatcagtt
     1321 tgaattattc accgacatgg aggcccagaa taccctcctt gacagtcttg acgtgcgcag
     1381 ctcaggggca tgatgtgact gtcgcccgta catttagccc atacatcccc atgtataatc
     1441 atttgcatcc atacattttg atggccgcac ggcgcgaagc aaaaattacg gctcctcgct
     1501 gcagacctgc gagcagggaa acgctcccct cacagacgcg ttgaattgtc cccacgccgc
     1561 gcccctgtag agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact
     1621 tccttttaaa atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg
     1681 ggtaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     1741 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     1801 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     1861 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     1921 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     1981 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     2041 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     2101 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     2161 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     2221 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     2281 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     2341 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     2401 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     2461 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     2521 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     2581 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     2641 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     2701 gaataatcag tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt
     2761 ttttttatat tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc
     2821 gcctcgacat catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca
     2881 atcgtatgtg aatgctggtc gctatactgc tgtcgattcg atactaacgc cgccatccat
     2941 gattctcgac tgtatttcta gctttgtatt tatgacattt ttcatagcta atacaaaagt
     3001 aagttaataa tatatttatc tgaatgctta tacatattaa aaatgaaaca taaaattggt
     3061 ataatgagga cttgtttgca aggttgatga aatttaactg tatttcttat attaatgtga
     3121 ttcttagtta taccaaataa attattttaa aataaaaacc aaaaccagta ccatttttga
     3181 ttgaatatgg gcatgtctag agtaagaaat tagcggagaa aaaataggac atgtcacata
     3241 gaaagggtat ttaaaaaaaa ataaaggaat tcaaaaggga gattcaccca acattacggt
     3301 tgaagattcg tagtctgtgt cttcttcttc ctcgtcatca tccaaacctg gaatttgttg
     3361 ccattcgtta taaaatggtt gaacaattat agaagacttc gtaagccggt aaggatcaaa
     3421 gggaaaatag gcatccatag tgtcgaaccc ttcctcgagc ggccgcatcg tgactgactg
     3481 acgatctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     3541 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     3601 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     3661 gtgtataatt cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     3721 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3781 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3841 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3901 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3961 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     4021 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     4081 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
     4141 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     4201 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4261 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     4321 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4381 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     4441 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4501 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4561 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4621 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4681 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4741 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4801 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4861 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4921 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4981 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5041 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5101 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5161 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5221 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5281 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5341 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5401 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5461 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5521 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5581 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5641 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     5701 cttacgcatc tgtgcggtat ttcacaccgc ataaattccg acaccatcga atggtgcaaa
     5761 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     5821 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     5881 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     5941 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     6001 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     6061 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     6121 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     6181 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     6241 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     6301 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     6361 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     6421 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     6481 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     6541 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     6601 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
     6661 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     6721 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
     6781 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
     6841 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     6901 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     6961 gcttccggct cgtatgttgt gt
//
