LOCUS       Exported                7450 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  A guide RNA targeting ACE2 in a lentiviral plasmid co-expressing 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    ACE2 g3
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  JOURNAL   Unpublished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 May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7450
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             1..181
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    228..353
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    846..1079
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1264..1308
                     /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        1486..1726
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1486..1506
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1657..1676
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    1900..2017
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2086..2297
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2317..2326
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2326..2349
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2350..3060
                     /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(2495..2513)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2763..2782
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    3076..3664
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3129..3149)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3547..3558)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3736..3969
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4047..4168
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4084..4103)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4138..4157
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4208..4343
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4270..4289
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4363..4382)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4364..4382)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4392..4409)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4392..4408)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4401..4423)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4550..5005
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4637..4656)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4847..4868
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5031..5135
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5136..5996
                     /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(5354..5373)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6167..6755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6656..6675
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6909..6926
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7043..7064
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7079..7109
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7117..7133
                     /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     7122..7144
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7141..7157
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7141..7157
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7176..7196
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7178..7196
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7224..7450
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaggg cctatttccc
     1501 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
     1561 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     1621 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     1681 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtgtca
     1741 agcagctaaa catgagttta agagctatgc tggaaacagc atagcaagtt taaataaggc
     1801 tagtccgtta tcaactttgc tggaaacagc aaagtggcac cgagtcggtg ctttttttga
     1861 attctagatc ttgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     1921 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     1981 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2041 agatccactt tggcgccggc tcgagtggct ccggtgcccg tcagtgggca gagcgcacat
     2101 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa
     2161 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
     2221 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
     2281 ttgccgccag aacacaggtg tcgtgacgcg ggatccgcca ccatggatta caaagacgat
     2341 gacgataaga tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
     2401 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     2461 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     2521 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
     2581 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     2641 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     2701 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     2761 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     2821 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     2881 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     2941 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     3001 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtag
     3061 acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     3121 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3181 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     3241 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     3301 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     3361 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     3421 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     3481 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     3541 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     3601 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     3661 ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     3721 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     3781 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     3841 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     3901 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     3961 tctctagcag tacgtatagt agttcatgtc atcttattat tcagtattta taacttgcaa
     4021 agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat
     4081 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4141 gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac
     4201 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4261 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     4321 gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat
     4381 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     4441 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     4501 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     4561 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4621 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4681 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4741 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     4801 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4861 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     4921 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4981 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5041 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5101 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5161 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5221 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5281 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5341 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5401 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5461 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5521 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5581 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     5641 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     5701 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     5761 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     5821 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     5881 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     5941 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6001 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6061 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6121 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6181 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6241 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6301 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6361 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6421 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6481 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6541 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6601 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6661 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6721 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6781 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     6841 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     6901 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     6961 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     7021 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     7081 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     7141 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
     7201 aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat
     7261 ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt
     7321 ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg
     7381 gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg
     7441 atacataaac
//
