LOCUS       sequence-178579-        7041 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP-CHMP7 in mammalian cells under a crippled promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7041)
  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 7041)
  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: pMGF182
FEATURES             Location/Qualifiers
     source          1..7041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        257..308
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     primer_bind     259..279
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     353..372
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        353..371
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(431..433,434..436,437..1147)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(431..433,434..436,437..1147)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(476..497)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(737..756)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1084..1105
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2558..2577)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     primer_bind     complement(2632..2649)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2638..2862
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2908..3336
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2995..3014)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3205..3226
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3345..3365)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3350..3679
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3530..3665
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3746..4540
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3800..3819)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4410..4429
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4714..4835
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4751..4770)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4805..4824
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4884..4900)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4884..4900)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4897..4919)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4908..4924
                     /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(4932..4938,4939..4956,4957..4962))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4977..4998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5115..5132)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5286..5874)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5366..5385)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6045..6836,6837..6905))
                     /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     6668..6687
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6906..7010)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgtcgta
      241 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      301 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
      361 ctcactatag ggagacccaa gctggctagc gtttaaacgg gccctctaga attatcgatc
      421 cggaggtacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
      481 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
      541 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
      601 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
      661 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
      721 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
      781 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
      841 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
      901 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
      961 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1021 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     1081 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     1141 gtacaaggga ggaacgggcg gtagtggagg cactggttgg tccccggagc gggaggccga
     1201 ggccccagcc gggggagacc cggcgggcct tctgcccccc gagtgggagg aggacgagga
     1261 gcgcatgtcc ttcctgttct ccgctttcaa gaggagtcgc gaggtgaaca gcaccgactg
     1321 ggacagcaag atgggcttct gggcgccgtt ggtgctgagc cacagccgcc gccagggggt
     1381 ggtgcgcctg cgtctgcggg acttgcagga ggcctttcag cgcaagggga gcgtcccgct
     1441 ggggctggcc acggtgctgc aggacctgct gcgtcgaggg gagctgcagc gggagtcaga
     1501 cttcatggcc agtgtagaca gcagctggat ctcctggggg gttggggtct tcctgctgaa
     1561 gcctctcaag tggactcttt ctaacatgct gggagataat aaggttccag ctgaggaggt
     1621 ccttgtcgct gtggagctgt tgaaggaaaa ggctgaggag gtgtatcgtc tgtatcagaa
     1681 ctcgcccctc tcctcccacc ccgtggtggc cctgtcagag ctcagcaccc tctgtgctaa
     1741 ctcctgccca gatgagagga ccttctactt ggtgttgctg cagctgcaga aggagaagag
     1801 ggtcacagtc ctcgagcaga acggggagaa gattgtgaag tttgcccgag ggccacgtgc
     1861 caaggtctct ccagtcaatg acgtagatgt tggggtgtac cagctgatgc agagtgaaca
     1921 gcttctctca cgcaaagtgg agtccttatc ccaggaagca gagaggtgta aagaagaagc
     1981 ccgccgggca tgccgagcag gaaagaagca gctggcactg aggtctctca aggccaagca
     2041 acggacagag aagcgcatcg aggccttgca tgccaagctg gacactgttc aaggcatcct
     2101 ggaccggatc tatgcctccc agacagatca gatggttttt aacgcctacc aggctggggt
     2161 aggagcactc aaactctcca tgaaggatgt cacagtggag aaggcagaga gcctcgtgga
     2221 tcagatccaa gagctctgtg acacccagga tgaagtttct cagactctgg ctggtggggt
     2281 aacaaatggc ttagattttg acagtgaaga actggagaag gaattggaca tcctccttca
     2341 ggataccacc aaagaacctt tggatctgcc tgacaacccc cgcaataggc attttaccaa
     2401 cagcgtgcct aaccctagga tctcagatgc tgaacttgaa gctgaacttg agaaactgtc
     2461 cttatcagag ggaggtttgg tcccaagcag taaatctcca aaaaggcaat tggaaccgac
     2521 tctaaagcca ttgtgactcg agaagcttgc tagcagatct ttttccctct gccaaaaatt
     2581 atggggacat catgaagccc cttgagcact taagtttaaa ccgctgatca gcctcgactg
     2641 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2701 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2761 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2821 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     2881 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     2941 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3001 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3061 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3121 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3181 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3241 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3301 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3361 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3421 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3481 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3541 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3601 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     3661 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     3721 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3781 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3841 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3901 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     3961 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4021 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4081 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4141 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4201 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4261 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4321 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4381 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4441 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4501 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4561 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4621 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4681 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4741 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4801 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4861 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4921 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4981 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5041 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5101 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5161 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5221 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5281 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5341 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5401 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5461 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5521 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5581 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5641 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5701 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5761 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5821 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5881 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5941 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6001 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6061 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6121 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6181 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6241 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6301 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6361 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6421 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6481 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6541 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6601 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6661 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6721 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6781 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6841 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6901 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6961 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7021 cgaaaagtgc cacctgacgt c
//
