LOCUS       sequence-462753-        6125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid to produce miniENVLPE particles.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6125)
  AUTHORS   Geilenkeuser J, Armbrust N, Steinmassl E, Du SW, Schmidt S, Binder 
            EMH, Li Y, Warsing NW, Wendel SV, von der Linde F, Schiele EM, Niu 
            X, Stroppel L, Berezin O, Santl TH, Orschmann T, Nelson K, Gruber C,
            Palczewska G, Menezes CR, Risaliti E, Engfer ZJ, Koleci N, Schmidts 
            A, Geerlof A, Palczewski K, Westmeyer GG, Truong DJ
  TITLE     Engineered nucleocytosolic vehicles for loading of programmable 
            editors.
  JOURNAL   Cell. 2025 May 15;188(10):2637-2655.e31. doi: 
            10.1016/j.cell.2025.03.015. Epub 2025 Apr 9.
  PUBMED    40209705
REFERENCE   2  (bases 1 to 6125)
  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: 
            "10.1016/j.cell.2025.03.015"; journalName: "Cell"; date: 
            "2025-05-15- 15"; volume: "188"; issue: "10"; pages: "2637-2655.e31"
COMMENT     Sequence Label: pCMV_miniENVLPE
FEATURES             Location/Qualifiers
     source          1..6125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      489..1077
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     978..997
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1231..1248
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1365..1386
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1401..1406,1407..1424,1425..1431)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1439..1455
                     /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     1444..1466
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1463..1479
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1463..1479
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        1503..1882
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1883..2086
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2037..2057
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2083..2107
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          2198..2770
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     primer_bind     2825..2844
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2825..2843
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3682..3738
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             3814..3840
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             3853..3879
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             3889..3915
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             3913..3936
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4507..4523)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4537..4554)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4543..4767
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4839..4856)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4839..4855)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4848..4870)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4996..5451)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5133..5154)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5345..5364
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5478..5582
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5583..5651,5652..6125,1..318)
                     /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     complement(5801..5820)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
       61 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
      121 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
      181 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
      241 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
      301 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
      361 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
      421 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
      481 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
      541 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
      601 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
      661 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
      721 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
      781 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
      841 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
      901 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
      961 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     1021 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     1081 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     1141 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     1201 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     1261 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     1321 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     1381 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     1441 gtgagcggat aacaatttca cacaggaaac agctatgacc atgaggcgcg ccccgccatg
     1501 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     1561 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     1621 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     1681 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     1741 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     1801 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     1861 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     1921 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     1981 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     2041 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg
     2101 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg
     2161 atccagcctc cggtcgaccg atcctgagaa cttcagggtg agtttgggga cccttgattg
     2221 ttctttcttt ttcgctattg taaaattcat gttatatgga gggggcaaag ttttcagggt
     2281 gttgtttaga atgggaagat gtcccttgta tcaccatgga ccctcatgat aattttgttt
     2341 ctttcacttt ctactctgtt gacaaccatt gtctcctctt attttctttt cattttctgt
     2401 aactttttcg ttaaacttta gcttgcattt gtaacgaatt tttaaattca cttttgttta
     2461 tttgtcagat tgtaagtact ttctctaatc actttttttt caaggcaatc agggtatatt
     2521 atattgtact tcagcacagt tttagagaac aattgttata attaaatgat aaggtagaat
     2581 atttctgcat ataaattctg gctggcgtgg aaatattctt attggtagaa acaactacac
     2641 cctggtcatc atcctgcctt tctctttatg gttacaatga tatacactgt ttgagatgag
     2701 gataaaatac tctgagtcca aaccgggccc ctctgctaac catgttcatg ccttcttctc
     2761 tttcctacag ctcctgggca acgtgctggt tgttgtgctg tctcatcatt ttggcattaa
     2821 ttaataatac gactcactat aggggccgcc accatgcacg gcctgcagga cgaccctgat
     2881 ctgcaggccc tgctgaaggg cagccagctg ctgaaagtga agtccagcag ctggcggcgg
     2941 gaaagattct acaagctgca agaggactgc aagaccatct ggcaggagtc tagaaaggtg
     3001 atgcgctccc cagaaagcca actgttcagc atcgaggaca tccaggaggt gcggatgggc
     3061 cacagaacag agggactgga aaagttcgcc agagatatcc ccgaggatag atgttttagc
     3121 attgtgttca aggaccagcg gaacaccctg gacctgatcg cccctagccc tgctgatgcc
     3181 cagcattggg tccagggcct cagaaagatc atccaccaca gcggctctat ggaccagaga
     3241 cagaaactgc agaccggtgc tagagcatct gttctgtctg gcggagcaca gcaggccgct
     3301 gctgatacag gccactctaa tcaggtgtcc cagaactaca gccccacctc catcctggac
     3361 atcagacagg gccccaaaga gcccttccgg gactacgtgg acagattcta caagaccctg
     3421 agagccgagc aggccagcca agaagtgaag aactggatga ccgagacact gctggtgcag
     3481 aacgccaatc ctgactgcaa gaccatcctg aaggctctgg gacctgccgc cacactggaa
     3541 gagatgatga cagcctgtca aggcgttggc ggccctggac acaaagctag agttctggcc
     3601 gaggccatga gccaagtgac caactccgct accatcatgg gaggatccgg cggcagaatg
     3661 aagcagctgg aagataaggt ggaagaactg ctgagcaaga actaccacct ggaaaacgag
     3721 gtggcccggc tgaagaagct cgttggagaa agaggtggca gcggcggagt ttctggatgg
     3781 cggctgttca agaagatcag cggcggttcc ggacctgctg ccaaaagagt gaaactggac
     3841 tccggactgc aactgcctcc actggaaaga ctgacactgg gagctgcccc agccgccaag
     3901 aagaaaaagc tggactacaa ggacgacgac gacaagggcg gagctagctc taagacaatc
     3961 gtgctgtctg tgggcgaagc caccagaaca ctgaccgaga tccagagcac agccgacaga
     4021 cagatcttcg aggaaaaagt gggccctctc gtgggcagac tgagactgac agcttccctg
     4081 agacagaacg gcgccaagac cgcctacaga gtgaacctga agctggacca ggccgacgtg
     4141 gtggattctg gccttcctaa agtgcggtac acccaagtgt ggtcccacga cgtgaccatc
     4201 gtggccaatt ctaccgaggc cagcagaaag agcctgtacg acctgaccaa gagcctggtg
     4261 gccacaagcc aggtggaaga tctggtcgtg aacctggtgc ctctcggacg tggcggcagc
     4321 gctggcggac tccagagcag acctgaacct acagctccac cagaggaatc cttccgcagc
     4381 ggcgtggaaa ccacaacacc tccacagaag caagagccca tcgacaaaga gctgtaccct
     4441 ctgaccagcc tgagaagcct gttcggcaac gatcccagca gccagtaaaa cgcgtaaatg
     4501 attgcagatc cactagttct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     4561 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     4621 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     4681 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     4741 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
     4801 gatccactag ttctagcctc gaggctagag cggccgccac tggccgtcgt tttacaacgt
     4861 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     4921 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     4981 ctgaatggcg aatggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     5041 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     5101 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     5161 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     5221 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     5281 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     5341 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     5401 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     5461 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5521 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5581 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5641 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5701 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5761 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5821 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5881 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5941 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     6001 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6061 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6121 tgcct
//
