LOCUS       sequence-190572-        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"
     promoter        85..136
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     primer_bind     87..107
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     181..200
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        181..199
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(259..261,262..264,265..975)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(259..261,262..264,265..975)
                     /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(304..325)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(565..584)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     912..933
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2386..2405)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     primer_bind     complement(2460..2477)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2466..2690
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2736..3164
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2823..2842)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3033..3054
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3173..3193)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3178..3507
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3358..3493
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3574..4368
                     /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(3628..3647)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4238..4257
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4542..4663
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4579..4598)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4633..4652
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4712..4728)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4712..4728)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4725..4747)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4736..4752
                     /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(4760..4766,4767..4784,4785..4790))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4805..4826
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4943..4960)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5114..5702)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5194..5213)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5873..6664,6665..6733))
                     /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     6496..6515
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6734..6838)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6913..6932)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
       61 ttgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
      121 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
      181 taatacgact cactataggg agacccaagc tggctagcgt ttaaacgggc cctctagaat
      241 tatcgatccg gaggtaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      301 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      361 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      421 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
      481 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      541 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
      601 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
      661 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
      721 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
      781 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
      841 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
      901 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
      961 gacgagctgt acaagggagg aacgggcggt agtggaggca ctggttggtc cccggagcgg
     1021 gaggccgagg ccccagccgg gggagacccg gcgggccttc tgccccccga gtgggaggag
     1081 gacgaggagc gcatgtcctt cctgttctcc gctttcaaga ggagtcgcga ggtgaacagc
     1141 accgactggg acagcaagat gggcttctgg gcgccgttgg tgctgagcca cagccgccgc
     1201 cagggggtgg tgcgcctgcg tctgcgggac ttgcaggagg cctttcagcg caaggggagc
     1261 gtcccgctgg ggctggccac ggtgctgcag gacctgctgc gtcgagggga gctgcagcgg
     1321 gagtcagact tcatggccag tgtagacagc agctggatct cctggggggt tggggtcttc
     1381 ctgctgaagc ctctcaagtg gactctttct aacatgctgg gagataataa ggttccagct
     1441 gaggaggtcc ttgtcgctgt ggagctgttg aaggaaaagg ctgaggaggt gtatcgtctg
     1501 tatcagaact cgcccctctc ctcccacccc gtggtggccc tgtcagagct cagcaccctc
     1561 tgtgctaact cctgcccaga tgagaggacc ttctacttgg tgttgctgca gctgcagaag
     1621 gagaagaggg tcacagtcct cgagcagaac ggggagaaga ttgtgaagtt tgcccgaggg
     1681 ccacgtgcca aggtctctcc agtcaatgac gtagatgttg gggtgtacca gctgatgcag
     1741 agtgaacagc ttctctcacg caaagtggag tccttatccc aggaagcaga gaggtgtaaa
     1801 gaagaagccc gccgggcatg ccgagcagga aagaagcagc tggcactgag gtctctcaag
     1861 gccaagcaac ggacagagaa gcgcatcgag gccttgcatg ccaagctgga cactgttcaa
     1921 ggcatcctgg accggatcta tgcctcccag acagatcaga tggtttttaa cgcctaccag
     1981 gctggggtag gagcactcaa actctccatg aaggatgtca cagtggagaa ggcagagagc
     2041 ctcgtggatc agatccaaga gctctgtgac acccaggatg aagtttctca gactctggct
     2101 ggtggggtaa caaatggctt agattttgac agtgaagaac tggagaagga attggacatc
     2161 ctccttcagg ataccaccaa agaacctttg gatctgcctg acaacccccg caataggcat
     2221 tttaccaaca gcgtgcctaa ccctaggatc tcagatgctg aacttgaagc tgaacttgag
     2281 aaactgtcct tatcagaggg aggtttggtc ccaagcagta aatctccaaa aaggcaattg
     2341 gaaccgactc taaagccatt gtgactcgag aagcttgcta gcagatcttt ttccctctgc
     2401 caaaaattat ggggacatca tgaagcccct tgagcactta agtttaaacc gctgatcagc
     2461 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2521 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2581 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2641 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2701 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2761 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2821 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2881 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2941 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3001 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3061 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3121 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     3181 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3241 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3301 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3361 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3421 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3481 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3541 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3601 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3661 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3721 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3781 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3841 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3901 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3961 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4021 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     4081 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     4141 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4201 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4261 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4321 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4381 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4441 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4501 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4561 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4621 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4681 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4741 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4801 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4861 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4921 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4981 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5041 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5101 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5161 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5221 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5281 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5341 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5401 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5461 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5521 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5581 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5641 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5701 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5761 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5821 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5881 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5941 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6001 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6061 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6121 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6181 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6241 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6301 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6361 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6421 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6481 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6541 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6601 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6661 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6721 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6781 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6841 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     6901 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     6961 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     7021 cttgaccgac aattgcatga a
//
