LOCUS       sequence-298697-        7450 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 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  JOURNAL   Unpublished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            "Unpublished"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: TMPRSS2 g3
FEATURES             Location/Qualifiers
     source          1..7450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(112..130)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     380..399
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    693..1281
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(746..766)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1164..1175)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1353..1586
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1664..1785
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1701..1720)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1755..1774
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1825..1960
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1980..1999)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1981..1999)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2009..2026)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2009..2025)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2018..2040)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2167..2622
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2254..2273)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2464..2485
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2648..2752
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2753..2821,2822..3613)
                     /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(2971..2990)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3784..4372
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4273..4292
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4526..4543
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4660..4681
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4696..4701,4702..4719,4720..4726)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4734..4750
                     /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     4739..4761
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4758..4774
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4758..4774
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4793..4813
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4795..4813
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4841..5067
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5068..5248
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5295..5420
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5913..6146
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6331..6375
                     /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        6553..6801
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6553..6573
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6724..6743
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    6967..7084
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        7153..7364
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7384..7393
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7393..7416
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(7417..7450,1..677)
                     /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"
ORIGIN
        1 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt
       61 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
      121 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt
      181 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
      241 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
      301 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
      361 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct
      421 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc
      481 tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg
      541 tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg
      601 actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg
      661 acgagctgta caagtagacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg
      721 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
      781 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
      841 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
      901 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
      961 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     1021 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     1081 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     1141 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     1201 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     1261 ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga
     1321 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     1381 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     1441 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     1501 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     1561 ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca
     1621 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc
     1681 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     1741 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta
     1801 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     1861 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     1921 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc
     1981 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     2041 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     2101 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     2161 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2221 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2281 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2341 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2401 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2461 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2521 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2581 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     2641 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2701 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     2761 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     2821 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     2881 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     2941 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3001 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3061 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3121 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3181 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3241 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     3301 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     3361 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     3421 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     3481 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     3541 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     3601 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     3661 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3721 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3781 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3841 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3901 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     3961 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4021 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4081 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4141 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4201 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4261 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4321 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4381 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4441 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4501 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     4561 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     4621 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     4681 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     4741 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     4801 ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc
     4861 ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag
     4921 caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa
     4981 cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta
     5041 tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg
     5101 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     5161 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     5221 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag
     5281 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag
     5341 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga
     5401 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa
     5461 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca
     5521 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac
     5581 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat
     5641 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg
     5701 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg
     5761 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
     5821 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
     5881 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
     5941 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
     6001 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
     6061 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
     6121 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
     6181 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
     6241 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
     6301 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
     6361 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
     6421 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
     6481 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     6541 aggggaccca gagagggcct atttcccatg attccttcat atttgcatat acgatacaag
     6601 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
     6661 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     6721 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     6781 ttgtggaaag gacgaaacac cgttgttctt cgaagtgacc aggtttaaga gctatgctgg
     6841 aaacagcata gcaagtttaa ataaggctag tccgttatca actttgctgg aaacagcaaa
     6901 gtggcaccga gtcggtgctt tttttgaatt ctagatcttg agacaaatgg cagtattcat
     6961 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     7021 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     7081 ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg agtggctccg
     7141 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
     7201 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
     7261 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
     7321 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgacgcggga
     7381 tccgccacca tggattacaa agacgatgac gataagatgg tgagcaaggg cgaggaggat
     7441 aacatggcca
//
