LOCUS       sequence-296045-        7450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A guide RNA targeting ACE2 in a lentiviral plasmid co-expressing 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7450)
  TITLE     CRISPR plasmids to knock out SARS-CoV-2 host genes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7450)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: ACE2 g3
FEATURES             Location/Qualifiers
     source          1..7450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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        1717..1965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2131..2248
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2317..2528
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2548..2557
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2557..2580
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2581..3291
                     /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(2726..2744)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2994..3013
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    3307..3895
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3360..3380)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3778..3789)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3967..4200
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4278..4399
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4315..4334)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4369..4388
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4439..4574
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4594..4613)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4595..4613)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4623..4640)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4623..4639)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4632..4654)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4781..5236
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4868..4887)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5078..5099
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5262..5366
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5367..5435,5436..6227)
                     /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(5585..5604)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6398..6986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6887..6906
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7140..7157
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7274..7295
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7310..7315,7316..7333,7334..7340)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7348..7364
                     /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     7353..7375
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7372..7388
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7372..7388
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7407..7427
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7409..7427
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgtgtc aagcagctaa
     1981 acatgagttt aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt
     2041 atcaactttg ctggaaacag caaagtggca ccgagtcggt gctttttttg aattctagat
     2101 cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     2161 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     2221 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact
     2281 ttggcgccgg ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
     2341 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
     2401 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
     2461 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
     2521 gaacacaggt gtcgtgacgc gggatccgcc accatggatt acaaagacga tgacgataag
     2581 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     2641 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     2701 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     2761 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     2821 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     2881 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     2941 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     3001 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     3061 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     3121 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     3181 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     3241 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta agcgcgttaa
     3301 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     3361 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     3421 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     3481 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     3541 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     3601 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     3661 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     3721 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     3781 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     3841 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga
     3901 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     3961 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg
     4021 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     4081 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     4141 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     4201 gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     4261 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4321 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4381 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
     4441 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4501 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     4561 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     4621 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     4681 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     4741 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     4801 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4861 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4921 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4981 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5041 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5101 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     5161 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5221 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5281 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5341 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5401 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5461 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5521 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5581 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5641 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5701 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5761 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5821 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5881 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     5941 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6001 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6061 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6121 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6181 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6241 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6301 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6361 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6421 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6481 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6541 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6601 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6661 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6721 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6781 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6841 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6901 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6961 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7021 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     7081 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     7141 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     7201 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     7261 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     7321 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     7381 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
     7441 agctgcaagc
//
