LOCUS       sequence-457186-        6952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP tagged MLF2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6952)
  AUTHORS   Prophet SM, Rampello AJ, Niescier RF, Gentile JE, Mallik S, Koleske 
            AJ, Schlieker C
  TITLE     Atypical nuclear envelope condensates linked to neurological 
            disorders reveal nucleoporin-directed chaperone activities.
  JOURNAL   Nat Cell Biol. 2022 Nov;24(11):1630-1641. doi: 
            10.1038/s41556-022-01001-y. Epub 2022 Oct 27.
  PUBMED    36302970
REFERENCE   2  (bases 1 to 6952)
  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: 
            "10.1038/s41556-022-01001-y"; journalName: "Nat Cell Biol"; date: 
            "2022-11"; volume: "24"; issue: "11"; pages: "1630-1641"
COMMENT     Sequence Label: MLF2-GFP-3xFLAG
FEATURES             Location/Qualifiers
     source          1..6952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        161..540
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        541..744
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     695..715
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     789..808
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        789..807
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1643..1652
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1649..1651,1652..1654,1655..2365)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1649..1651,1652..1654,1655..2365)
                     /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(1694..1715)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1955..1974)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2302..2323
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2369..2392
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2402..2428
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2469..2486)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2475..2699
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2745..3173
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2832..2851)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3042..3063
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3182..3202)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3187..3516
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3367..3502
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3429..3448
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3583..4377
                     /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(3637..3656)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4247..4266
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4551..4672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4588..4607)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4642..4661
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4721..4737)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4721..4737)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4734..4756)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4745..4761
                     /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(4769..4775,4776..4793,4794..4799))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4814..4835
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4952..4969)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5123..5711)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5203..5222)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5882..6673,6674..6742))
                     /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     6505..6524
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6743..6847)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6922..6941)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
       61 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
      121 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
      181 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      241 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      301 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      361 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      421 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      481 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      541 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      601 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      661 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      721 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      781 atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt aaacttaagc
      841 ttggtaccga gctcggatcc actagtccag tgtggtggaa ttcgccacca tgttccgctt
      901 catgagggac gtggagcctg aggatcccat gttcctgatg gatccctttg ctattcaccg
      961 tcagcatatg agccgtatgt tgtcaggtgg ctttggatat agccccttcc tcagcatcac
     1021 agatggcaac atgccaggga ccaggcctgc cagccgccgg atgcagcagg ctggagctgt
     1081 ctcccccttt gggatgctgg gaatgtcggg tggtttcatg gacatgtttg ggatgatgaa
     1141 tgacatgatt ggaaacatgg aacacatgac agctggaggc aattgccaga ccttctcatc
     1201 ttccactgtc atctcctact ccaatacggg tgatggtgcc cccaaggtct accaagagac
     1261 atcagagatg cgctcggcac caggcgggat ccgggagaca cggaggactg ttcgggattc
     1321 agacagtgga ctggagcaga tgtccattgg gcatcacatc cgggacaggg ctcacatcct
     1381 ccagcgctcc cgaaaccatc gcacggggga ccaggaggag cggcaggact atatcaacct
     1441 ggatgagagt gaggccgcag cgtttgatga cgagtggcgg cgggagacct cccgattccg
     1501 gcagcagcgt cccctggagt ttcggcggct tgagtcctca ggggctgggg gacgaagggc
     1561 ggaggggcct ccccgcctgg ccatccaggg acctgaggac tccccttccc gacagtcccg
     1621 ccgctatgac tggggaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
     1681 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     1741 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     1801 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     1861 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     1921 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     1981 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2041 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     2101 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     2161 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     2221 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     2281 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     2341 tctcggcatg gacgagctgt acaagatgga ttacaaggat gacgacgata agtagctcga
     2401 gtatccgtac gacgtaccag actacgcact cgactaatct agagggcccg tttaaacccg
     2461 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2521 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2581 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2641 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2701 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2761 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2821 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2881 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2941 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3001 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3061 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3121 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     3181 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3241 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3301 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3361 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3421 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     3481 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3541 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     3601 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3661 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3721 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     3781 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3841 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3901 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3961 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     4021 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4081 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     4141 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4201 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4261 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4321 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     4381 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     4441 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4501 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4561 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4621 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4681 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4741 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4801 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4861 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4921 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4981 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5041 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5101 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5161 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5221 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5281 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5341 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5401 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5461 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5521 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5581 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5641 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5701 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5761 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5821 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5881 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5941 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6001 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6061 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6121 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6181 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6241 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6301 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6361 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6421 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6481 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6541 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6601 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6661 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6721 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6781 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6841 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     6901 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gc
//
