LOCUS       Exported                8608 bp ds-DNA     circular SYN 02-DEC-2021
DEFINITION  Expressing paired pegRNAs from human U6 and H1 promoters to make 
            710-bp deletion on e-NMU locus.
ACCESSION   .
VERSION     .
KEYWORDS    Lentiguide-Blast-eNMU710
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8608)
  AUTHORS   Choi J, Chen W, Suiter CC, Lee C, Chardon FM, Yang W, Leith A, Daza 
            RM, Martin B, Shendure J
  TITLE     Precise genomic deletions using paired prime editing.
  JOURNAL   Nat Biotechnol. 2021 Oct 14. pii: 10.1038/s41587-021-01025-z. doi: 
            10.1038/s41587-021-01025-z.
  PUBMED    34650269
REFERENCE   2  (bases 1 to 8608)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 2, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8608
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        29..59
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    67..83
                     /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     72..94
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     91..107
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     91..107
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     126..146
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        128..146
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        174..400
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             401..581
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    628..753
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1246..1479
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1664..1708
                     /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"
     promoter        1886..2126
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1886..1906
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2057..2076
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2156..2231
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     2298..2314
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2773..2890
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3029..4207
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3260..4198
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4155..4175
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4214..4223
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4220..4618
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    4634..5222
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4687..4707)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5105..5116)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5294..5527
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5605..5726
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5642..5661)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5696..5715
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5766..5901
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5828..5847
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(5921..5940)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5922..5940)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5950..5966)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      6108..6563
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6195..6214)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6405..6426
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6589..6693
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6694..7554
                     /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(6912..6931)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7725..8313
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8214..8233
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8467..8484
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8601..14
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
       61 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
      121 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc
      181 ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca
      241 aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt
      301 attaggaagg caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg
      361 cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca
      421 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
      481 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
      541 atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac
      601 ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg
      661 cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga
      721 ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg
      781 cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata
      841 gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca
      901 gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa
      961 cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata
     1021 aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc
     1081 gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag
     1141 aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa
     1201 ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct
     1261 tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca
     1321 ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga
     1381 ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat
     1441 cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg
     1501 aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga
     1561 acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag
     1621 cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt
     1681 attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg
     1741 gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc
     1801 tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca
     1861 cctcccaacc ccgaggggac ccagagaggg cctatttccc atgattcctt catatttgca
     1921 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat
     1981 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     2041 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     2101 ggctttatat atcttgtgga aaggacgaaa caccgaaggg gcatgaagtt tactggtttt
     2161 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     2221 cgagtcggtg caggtcagag tcctggctct gtgactcagt gataaacttc atgccttttt
     2281 ttcccagtca cgacgttgta aaacgacggc cagtgaattc gaacgctgac gtcatcaacc
     2341 cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa cacccagcgc gcgtgcgccc
     2401 tggcaggaag atggctgtga gggacagggg agtggcgccc tgcaatattt gcatgtcgct
     2461 atgtgttcyg ggaaatcacc ataaacgtga aatgtctttg gatttgggaa tcttataagt
     2521 tctgtatgag gaccacagat ccccagctct gtgactcagt gacctggaat agaaaacaaa
     2581 agtttaagtt attctaaggc cagtccggaa tcatcctaaa aaggaggcac cgagtcggtg
     2641 cacatggtac ccatgaaggg gcatgaagtt tatcactgag tcacattttt ttaagcttgg
     2701 gccgctcgag gtacctctct acaaagcttg gcgtaactag atcttgagac aaatggcagt
     2761 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     2821 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     2881 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgaggg
     2941 ggcccgggtg caaagatgga taaagtttta aacagagagg aatctttgca gctaatggac
     3001 cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac
     3061 atcgcccaca gtccccgaga agttgggggg aggggtctgc aattgatccg gtgcctagag
     3121 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     3181 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     3241 gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac
     3301 gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga
     3361 tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc
     3421 ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg
     3481 gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg
     3541 atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct
     3601 gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg
     3661 cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc
     3721 tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg
     3781 ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg
     3841 ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc
     3901 acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga
     3961 gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt
     4021 aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga
     4081 agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg
     4141 atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt
     4201 gtcgtgacgt acggccacca tggccaagcc tttgtctcaa gaagaatcca ccctcattga
     4261 aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc
     4321 agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg
     4381 accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac
     4441 ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg
     4501 acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca
     4561 gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaaac
     4621 gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     4681 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     4741 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     4801 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     4861 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     4921 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     4981 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     5041 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     5101 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     5161 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     5221 gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     5281 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct
     5341 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     5401 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     5461 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     5521 tctagcagta cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     5581 aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa
     5641 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5701 ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc
     5761 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     5821 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     5881 gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta
     5941 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     6001 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     6061 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     6121 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     6181 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     6241 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     6301 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     6361 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     6421 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     6481 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     6541 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     6601 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     6661 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6721 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6781 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6841 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6901 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6961 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     7021 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     7081 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     7141 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     7201 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     7261 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     7321 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     7381 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     7441 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     7501 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     7561 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     7621 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     7681 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7741 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7801 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7861 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7921 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7981 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     8041 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     8101 tacctacagc gtgagctacg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     8161 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     8221 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     8281 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     8341 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     8401 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     8461 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     8521 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     8581 ggcagtgagc gcaacgcaat taatgtga
//
