LOCUS       sequence-298678-        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  JOURNAL   Unpublished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            "Unpublished"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 g2
FEATURES             Location/Qualifiers
     source          1..7450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    449..682
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             867..911
                     /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        1089..1337
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1089..1109
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1260..1279
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    1503..1620
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1689..1900
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1920..1929
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1929..1952
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1953..2663
                     /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(2098..2116)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2366..2385
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2679..3267
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2732..2752)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3150..3161)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3339..3572
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3650..3771
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3687..3706)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3741..3760
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3811..3946
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3966..3985)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3967..3985)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3995..4012)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3995..4011)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4004..4026)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4153..4608
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4240..4259)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4450..4471
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4634..4738
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4739..4807,4808..5599)
                     /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(4957..4976)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5770..6358
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6259..6278
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6512..6529
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6646..6667
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6682..6687,6688..6705,6706..6712)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6720..6736
                     /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     6725..6747
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6744..6760
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6744..6760
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6779..6799
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6781..6799
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6827..7053
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             7054..7234
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7281..7406
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
ORIGIN
        1 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
       61 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
      121 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
      181 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
      241 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc
      301 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
      361 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
      421 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
      481 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
      541 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
      601 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
      661 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
      721 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
      781 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
      841 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
      901 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
      961 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
     1021 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
     1081 gacccagaga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     1141 ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     1201 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     1261 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     1321 ggaaaggacg aaacaccgaa acatggaaca gagatgcggt ttaagagcta tgctggaaac
     1381 agcatagcaa gtttaaataa ggctagtccg ttatcaactt tgctggaaac agcaaagtgg
     1441 caccgagtcg gtgctttttt tgaattctag atcttgagac aaatggcagt attcatccac
     1501 aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     1561 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     1621 cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgagtg gctccggtgc
     1681 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     1741 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     1801 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     1861 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac gcgggatccg
     1921 ccaccatgga ttacaaagac gatgacgata agatggtgag caagggcgag gaggataaca
     1981 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
     2041 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     2101 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     2161 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     2221 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     2281 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     2341 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     2401 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     2461 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     2521 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     2581 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     2641 gcatggacga gctgtacaag tagacgcgtt aagtcgacaa tcaacctctg gattacaaaa
     2701 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     2761 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     2821 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     2881 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     2941 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3001 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3061 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     3121 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     3181 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     3241 ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc
     3301 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     3361 acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc
     3421 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     3481 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     3541 acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat
     3601 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat
     3661 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     3721 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     3781 gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3841 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3901 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca
     3961 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
     4021 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     4081 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     4141 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4201 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4261 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4321 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     4381 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4441 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     4501 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4561 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     4621 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4681 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4741 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4801 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4861 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4921 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4981 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5041 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5101 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5161 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5221 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5281 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5341 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5401 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5461 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5521 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5581 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5641 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5701 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5761 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5821 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5881 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5941 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6001 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6061 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6121 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6181 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6241 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6301 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6361 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6421 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6481 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     6541 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     6601 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     6661 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     6721 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc
     6781 aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca
     6841 atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag
     6901 aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga
     6961 aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat
     7021 attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga
     7081 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     7141 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     7201 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
     7261 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
     7321 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
     7381 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
     7441 aaaaaattcg
//
