LOCUS       sequence-298699-        7449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A guide RNA targeting TMPRSS2 in a lentiviral plasmid co-expressing 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7449)
  TITLE     CRISPR plasmids to knock out SARS-CoV-2 host genes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7449)
  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            "Unpublished"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: TMPRSS2 g2
FEATURES             Location/Qualifiers
     source          1..7449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /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        1713..1961
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1713..1733
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1884..1903
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2126..2243
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2312..2523
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2543..2552
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2552..2575
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2576..3286
                     /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(2721..2739)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2989..3008
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    3302..3890
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3355..3375)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3773..3784)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3962..4195
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4273..4394
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4310..4329)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4364..4383
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4434..4569
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4589..4608)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4590..4608)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4618..4635)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4618..4634)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4627..4649)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4776..5231
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4863..4882)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5073..5094
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5257..5361
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5362..5430,5431..6222)
                     /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(5580..5599)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6393..6981
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6882..6901
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7135..7152
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7269..7290
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7305..7310,7311..7328,7329..7335)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7343..7359
                     /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     7348..7370
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7367..7383
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7367..7383
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7402..7422
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7404..7422
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggaccca gagagggcct atttcccatg attccttcat
     1741 atttgcatat acgatacaag gctgttagag agataattag aattaatttg actgtaaaca
     1801 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     1861 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     1921 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggggaacgt cgattcttgc
     1981 cgtttaagag ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa
     2041 ctttgctgga aacagcaaag tggcaccgag tcggtgcttt ttttgaattc tagatcttga
     2101 gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
     2161 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
     2221 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc
     2281 gccggctcga gtggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
     2341 gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta
     2401 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     2461 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     2521 caggtgtcgt gacgcgggat ccgccaccat ggattacaaa gacgatgacg ataagatggt
     2581 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     2641 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     2701 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     2761 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     2821 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     2881 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     2941 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     3001 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     3061 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     3121 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     3181 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     3241 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtagacgc gttaagtcga
     3301 caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     3361 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     3421 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     3481 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     3541 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     3601 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     3661 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     3721 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     3781 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     3841 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta
     3901 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     3961 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct
     4021 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     4081 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     4141 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg
     4201 tatagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca
     4261 gagagtgaga ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4321 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4381 atcaatgtat cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc
     4441 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4501 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     4561 ttggaggcct agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     4621 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     4681 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4741 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     4801 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     4861 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     4921 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     4981 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5041 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5101 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     5161 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5221 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5281 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5341 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5401 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5461 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5521 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5581 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5641 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5701 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5761 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5821 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5881 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     5941 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6001 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6061 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6121 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6181 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6241 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6301 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6361 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6421 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6481 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6541 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6601 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6661 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6721 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6781 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6841 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6901 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6961 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7021 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     7081 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     7141 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     7201 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     7261 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     7321 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     7381 gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg
     7441 caagcttaa
//
