LOCUS       sequence-190617-        8096 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses LEMD2-mCherry in mammalian cells under a crippled 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8096)
  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 8096)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pMGF196
FEATURES             Location/Qualifiers
     source          1..8096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        147..198
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     primer_bind     149..169
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     243..262
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        243..261
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1860..2570
                     /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(2005..2023)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2273..2292
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2592..2611)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     primer_bind     complement(2666..2683)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2672..2896
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2942..3370
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3029..3048)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3239..3260
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3379..3399)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3384..3713
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3564..3699
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3626..3645
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     LTR             3792..4217
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     CDS             complement(4398..4997)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4481..4501)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4978..4997
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(5066..5085)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     polyA_signal    5659..5780
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5696..5715)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5750..5769
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5829..5845)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5829..5845)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5842..5864)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5853..5869
                     /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(5877..5883,5884..5901,5902..5907))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5922..5943
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6060..6077)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6231..6819)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6311..6330)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6990..7781,7782..7850))
                     /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     7613..7632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7851..7955)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8030..8049)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
       61 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      121 cgttgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
      181 gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa
      241 attaatacga ctcactatag ggagacccaa gctggctagc gtttaaacgg gccctctaga
      301 attatcgatc cggaggtacc atggccggcc tgtcggacct ggaactgcgg cgggagctgc
      361 aggccctggg cttccagcca ggacccatca ccgacaccac ccgggatgtc taccgcaaca
      421 agctgcgccg cctgcggggc gaggcccggc tgcgcgacga ggagcggctg cgggaggagg
      481 cccggccgcg gggcgaggag cggttacggg aagaggcccg gttacgcgag gatgcgccgc
      541 tgcgcgcccg gcccgccgcg gcctctccgc gggcggagcc ctggctctcc cagccggcct
      601 cgggctcggc ctacgcgacc cctggggcct acggtgatat ccggccctcc gcggcttcct
      661 gggtagggag ccgcggcctc gcctatcctg cccgcccggc gcaactcagg cgccgcgcct
      721 cggtccgggg cagctccgag gaggacgagg acgcccggac gcccgacagg gccacgcagg
      781 gcccgggtct cgcggcccgc cgctggtggg cagcgtctcc cgccccggcg cggctgcctt
      841 cctccctcct cggtcccgac ccgcgcccgg gcctgcgggc gactcgagcg ggccctgctg
      901 gcgcggcgag ggcccggcct gaggtggggc gccggctgga gcgctggctc tctcggcttc
      961 tgctctgggc cagcctaggg ctactgctcg tcttcctggg catcctttgg gtgaagatgg
     1021 gcaagccctc agcgccgcag gaggcggagg acaacatgaa gttattgcca gtggactgtg
     1081 agagaaaaac agatgagttc tgtcaggcca agcagaaggc agccttgctg gagctgctgc
     1141 atgaactcta caatttcctg gccatccaag ctggtaattt tgagtgtgga aatccagaga
     1201 atctaaaaag caaatgcatt cctgttatgg aagcccaaga atatatagcc aatgtgacca
     1261 gcagctcctc cgccaagttt gaagccgcac tgacctggat actgagcagt aacaaggacg
     1321 tgggcatctg gttgaaagga gaagaccagt ctgaattggt gacgactgtg gacaaggtgg
     1381 tctgcctgga atctgcccac ccccgcatgg gtgttggctg ccgcctgagc cgggccttgc
     1441 tcactgctgt caccaacgtg ctcatcttct tctggtgctt ggcttttttg tgggggctcc
     1501 taattctcct aaaatatcgg tggcgaaagt tagaagagga ggaacaagcc atgtatgaga
     1561 tggtgaagaa gattatagac gtggtccagg accattacgt ggactgggag caggacatgg
     1621 agcgctatcc atatgtaggc atcctgcacg tgcgcgacag cttgatccct ccacagagcc
     1681 ggaggcgcat gaagcgtgtc tgggaccgag ctgtggagtt cctggcctcc aacgaatccc
     1741 ggatccagac ggagtcccac cgcgttgcag gagaggacat gctggtgtgg agatggacta
     1801 agccctcttc cttctctgac tcagagcgag gaggaacggg cggtagtgga ggcactggta
     1861 tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg
     1921 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     1981 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct
     2041 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc
     2101 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg
     2161 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
     2221 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
     2281 tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg
     2341 ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg
     2401 aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca
     2461 acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac
     2521 gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtga ctcgagaagc
     2581 ttgctagcag atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag
     2641 cacttaagtt taaaccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2701 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2761 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2821 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2881 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     2941 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3001 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3061 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3121 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3181 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3241 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3301 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3361 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     3421 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3481 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3541 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3601 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3661 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3721 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     3781 tgcactacgt gaatgaaaga cccccgctga cgggtagtca atcactcaga ggagaccctc
     3841 ccaaggaaca gcgagaccac aagtcggatg caactgcaag agggtttatt ggatacacgg
     3901 gtacccgggc gactcagtca atcggaggac tggcgccccg agtgaggggt tgtgggctct
     3961 tttattgagc tcggggagca gaagcgcgcg aacagaagcg agaagcgaac tgattggtta
     4021 gttcaaataa ggcacagggt catttcaggt ccttggggca ccctggaaac atctgatggt
     4081 tctctagggt cgaccctgtt ccatctgttc ctgaccttga tctgaacttc tctattctca
     4141 gttatgtatt tttccatgcc ttgcaaaatg gcgttactta agctagcttg ccaaacctac
     4201 aggtggggtc tttcattccc ccctttttct ggagactaaa taaaatcttt tattttatct
     4261 atggctcgta ctctataggc ttcagctggt gatattgttg agtcaaaact agagcctgga
     4321 ccactgatat ctcgagacgc cgatgcatgg ggtcgtgcgc tcctttcggt cgggcgctgc
     4381 gggtcgtggg gcgggcgtca ggcaccgggc ttgcgggtca tgcaccaggt gcgcggtcct
     4441 tcgggcacct cgacgtcggc ggtgacggtg aagccgagcc gctcgtagaa ggggaggttg
     4501 cggggcgcgg aggtctccag gaaggcgggc accccggcgc gctcggccgc ctccactccg
     4561 gggagcacga cggcgctgcc cagacccttg ccctggtggt cgggcgagac gccgacggtg
     4621 gccaggaacc acgcgggctc cttgggccgg tgcggcgcca ggaggccttc catctgttgc
     4681 tgcgcggcca gccgggaacc gctcaactcg gccatgcgcg ggccgatctc ggcgaacacc
     4741 gcccccgctt cgacgctctc cggcgtggtc cagaccgcca ccgcggcgcc gtcgtccgcg
     4801 acccacacct tgccgatgtc gagcccgacg cgcgtgagga agagttcttg cagctcggtg
     4861 acccgctcga tgtggcggtc cggatcgacg gtgtggcgcg tggcggggta gtcggcgaac
     4921 gcggcggcga gggtgcgtac ggccctgggg acgtcgtcgc gggtggcgag gcgcaccgtg
     4981 ggcttgtact cggtcatggt aagcttgggc tgcaggtcga aaggcccgga gatgaggaag
     5041 aggagaacag cgcggcagac gtgcgctttt gaagcgtgca gaatgccggg cctccggagg
     5101 accttcgggc gcccgccccg cccctgagcc cgcccctgag cccgcccccg gacccacccc
     5161 ttcccagcct ctgagcccag aaagcgaagg agcaaagctg ctattggccg ctgccccaaa
     5221 ggcctacccg cttccattgc tcagcggtgc tgtccatctg cacgagacta gtgagacgtg
     5281 ctacttccat ttgtcacgtc ctgcacgacg cgagctgcgg ggcggggggg aacttcctga
     5341 ctaggggagg agtagaaggt ggcgcgaagg ggccaccaaa gaacggagcc ggttggcgcc
     5401 taccggtgga tgtggaatgt gtgcgaggcc agaggccact tgtgtagcgc caagtgccca
     5461 gcgggggtct ttcattcacg tagtggcggg actctggggt tcgaaatgac cgaccaagcg
     5521 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     5581 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     5641 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     5701 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     5761 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     5821 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5881 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5941 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6001 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     6061 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     6121 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     6181 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6241 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6301 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6361 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6421 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6481 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6541 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6601 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6661 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6721 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6781 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6841 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6901 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6961 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     7021 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     7081 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     7141 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7201 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7261 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7321 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7381 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7441 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7501 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7561 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7621 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7681 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7741 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7801 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7861 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7921 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7981 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     8041 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgct
//
