LOCUS       sequence-453883-        7788 bp    DNA     circular SYN 14-APR-2025
DEFINITION  miniEDV production plasmid. Expresses the miniGag (CAG promoter) and
            sgRNA (human U6 promoter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7788)
  AUTHORS   Ngo W, Peukes J, Baldwin A, Xue ZW, Hwang S, Stickels RR, Lin Z, 
            Satpathy AT, Wells JA, Schekman R, Nogales E, Doudna JA
  TITLE     Mechanism-guided engineering of a minimal biological particle for 
            genome editing.
  JOURNAL   Proc Natl Acad Sci U S A. 2025 Jan 7;122(1):e2413519121. doi: 
            10.1073/pnas.2413519121. Epub 2024 Dec 30.
  PUBMED    39793042
REFERENCE   2  (bases 1 to 7788)
  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"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2025-01-7"; volume: "122(1):e2413519121. 
            doi"; pages: " 10.1073/pnas.2413519121. Epub 2024 Dec 30"
COMMENT     Sequence Label: pWN_U6-B2M-miniGag
FEATURES             Location/Qualifiers
     source          1..7788
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(473..1264,1265..1333))
                     /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     1096..1115
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1334..1438)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1522..1770
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1522..1542
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1693..1712
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1924..2303
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2305..2582
                     /label=chicken beta-actin promoter
     intron          2583..3599
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3523..3545
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3607..3626
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     misc_feature    5207..5440
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5625..5669
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     CDS             5963..5989
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(6274..6293)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6339..6394
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6393..6412)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6755..6771)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6755..6771)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6768..6790)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6779..6795
                     /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(6803..6809,6810..6827,6828..6833))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6848..6869
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6928..7123
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6974..7109
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    7129..7263
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7166..7185)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7220..7239
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7331..7348)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(7502..7788,1..302))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7582..7601)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
       61 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      121 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
      181 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      241 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
      301 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
      361 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
      421 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
      481 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
      541 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
      601 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
      661 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
      721 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
      781 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
      841 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
      901 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
      961 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     1021 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     1081 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     1141 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     1201 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     1261 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     1321 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     1381 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     1441 catttccccg aaaagtgcca cctgggtcga ccgggtttat tacagggaca gcagagatcc
     1501 agtttggtta attaaggtac cgagggccta tttcccatga ttccttcata tttgcatata
     1561 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     1621 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     1681 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     1741 ttatatatct tgtggaaagg acgaaacacc ggagtagcgc gagcacagct agtttaagag
     1801 ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
     1861 tggcaccgag tcggtgcttt ttttgaattc gctagctagg tcttgaaagg agtgggaatt
     1921 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     1981 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2041 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     2101 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2161 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2221 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2281 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
     2341 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
     2401 cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc
     2461 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
     2521 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
     2581 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     2641 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     2701 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
     2761 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
     2821 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
     2881 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     2941 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     3001 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     3061 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     3121 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     3181 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
     3241 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     3301 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     3361 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     3421 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     3481 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     3541 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     3601 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttcgggccgc
     3661 gttgacgcgc acggcaagag gcgaggggcg gcgactggtg agagatgggt gcgagagcgt
     3721 cagtattaag cgggggagaa ttagatcgat gggaaaaaat tcggttaagg ccagggggaa
     3781 agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag
     3841 ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac
     3901 catcccttca gacaggatca gaagaactta gatcattata taatacagta gcaaccctct
     3961 attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg
     4021 aagagcaaaa caaaagtaag aaaaaagcac agcaagcagc agctgacaca ggacacagca
     4081 atcaggtcag ccaaaattac cctatagtgc agagcattct ggacataaga caaggaccaa
     4141 aggaaccctt tagagactat gtagaccgat tctataaaac tctaagagcc gagcaagctt
     4201 cacaagaggt aaaaaattgg atgacagaaa ccttgttggt ccaaaatgcg aacccagatt
     4261 gtaagactat tttaaaagca ttgggaccag gagcgacact agaagaaatg atgacagcat
     4321 gtcagggagt ggggggaccc ggccataaag caagagtttt ggctgaagca atgagccaag
     4381 taacaaatcc agctaccata atgatacaga aaggcagaca ggctaatttt ttagggaaga
     4441 tctggccttc ccacaaggga aggccaggga attttcttca gagcagacca gagccaacag
     4501 ccccaccaga agagagcttc aggtttgggg aagagacaac aactccctct cagaagcagg
     4561 agccgataga caaggaactg tatcctttag cttccctcag atcactcttt ggcagcgacc
     4621 cctcgtcaca ataacacatg gaattctgca acaactgctg tttatccatt tcagaattgg
     4681 gttcgacata gcagaatagg cgttactcga cagaggagag caagaaatgg agccagtaga
     4741 tcctagacta gagccctgga agcatccagg aagtcagcct aaaactgctt gtaccaattg
     4801 ctattgtaaa aagtgttgct ttcattgcca agtttgtttc atgacaaaag ccttaggcat
     4861 ctcctatggc aggaagaagc ggagacagcg acgaagagct catcagaaca gtcagactca
     4921 tcaagcttct ctatcaaagc agtaagtagt acatgtaatg caacctataa tagtagcaat
     4981 agtagcatta gtagtagcaa taataatagc aatagttgtg tggtccatag taatcataga
     5041 atataggaaa atggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     5101 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     5161 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     5221 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     5281 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     5341 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     5401 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     5461 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     5521 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     5581 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     5641 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     5701 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     5761 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     5821 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     5881 gagacagaga cagatccatt cgattagtga acggatcctt ggcacttatc tgggacgatc
     5941 tgcggagcct gtgcctcttc agctaccacc gcttgagaga cttactcttg attgtaacga
     6001 ggattgtgga acttctggga cgcagggggt gggaagccct caaatattgg tggaatctcc
     6061 tacaatattg gagtcaggag ctaaagaata gtgctgttag cttgctcaat gccacagcca
     6121 tagcagtagc tgaggggaca gatagggtta tagaagtagt acaaggagct tgtagagcta
     6181 ttcgccacat acctagaaga ataagacagg gcttggaaag gattttgcta taagctcgaa
     6241 acaaccggta cctctagaac tatagctagc agatcttttt ccctctgcca aaaattatgg
     6301 ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat
     6361 tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa
     6421 tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct
     6481 ggctgccatg aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc
     6541 tgtccattcc ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg
     6601 ttttgtgtta tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag
     6661 atttttcctc ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc
     6721 cctcgacctg cagcccaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     6781 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     6841 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     6901 cgggaaacct gtcgtgccag cggatccgca tctcaattag tcagcaacca tagtcccgcc
     6961 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     7021 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     7081 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctaa cttgtttatt
     7141 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     7201 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     7261 atccgctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     7321 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     7381 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     7441 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     7501 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     7561 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     7621 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     7681 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     7741 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcg
//
