LOCUS       sequence-265965-        8276 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression CasRx guideRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8276)
  TITLE     Unpublished
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8276)
  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            "Unpublished"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: pAK_lentiguide_DR30_Puro
FEATURES             Location/Qualifiers
     source          1..8276
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..1187
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          240..1178
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1135..1155
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1200..1799
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1200..1219)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1696..1716
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1815..2403
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1868..1888)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2286..2297)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2475..2708
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2786..2907
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2823..2842)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2877..2896
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2947..3082
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3102..3121)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3103..3121)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3131..3148)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3131..3147)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3140..3162)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3289..3744
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3376..3395)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3586..3607
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3770..3874
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3875..3943,3944..4735)
                     /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(4093..4112)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4906..5494
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5395..5414
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5648..5665
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5782..5803
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5818..5823,5824..5841,5842..5848)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5856..5872
                     /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     5861..5883
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5880..5896
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5880..5896
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5915..5935
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5917..5935
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5963..6189
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6190..6370
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6417..6542
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7035..7268
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7453..7497
                     /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        7675..7923
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7675..7695
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7846..7865
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7925..7954
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     misc_feature    8029..8146
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga
       61 agttgggggg aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact
      121 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata
      181 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg
      241 taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc
      301 cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt cgggttggaa
      361 gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg
      421 aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc
      481 tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct gcgacgcttt
      541 ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt atttcggttt
      601 ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg
      661 gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc cggcctgctc
      721 tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt
      781 cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca gggagctcaa
      841 aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaaaggg
      901 cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc
      961 acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt
     1021 atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca gcttggcact
     1081 tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc
     1141 ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgacgt acggccacca
     1201 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     1261 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     1321 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     1381 tcggcaaggt gtgggtcgcg gacgacggcg ccgccgtggc ggtctggacc acgccggaga
     1441 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     1501 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     1561 ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca
     1621 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     1681 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     1741 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa
     1801 cgcgttaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     1861 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     1921 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     1981 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     2041 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     2101 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     2161 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     2221 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     2281 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     2341 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     2401 cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     2461 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
     2521 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     2581 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     2641 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     2701 ctctagcagt acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa
     2761 gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata
     2821 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     2881 tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact
     2941 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3001 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     3061 tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt
     3121 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     3181 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     3241 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
     3301 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3361 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3421 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3481 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3541 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3601 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3661 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     3721 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     3781 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     3841 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     3901 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     3961 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     4021 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     4081 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     4141 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     4201 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     4261 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     4321 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     4381 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     4441 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     4501 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     4561 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     4621 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     4681 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     4741 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     4801 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     4861 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4921 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4981 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5041 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5101 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5161 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5221 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5281 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5341 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5401 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5461 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5521 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5581 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     5641 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     5701 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     5761 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     5821 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     5881 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
     5941 aaagctggag ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg
     6001 gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc atgccgattg
     6061 gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg tctgacatgg
     6121 attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg cctagctcga
     6181 tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     6241 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     6301 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     6361 atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct
     6421 ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
     6481 tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg
     6541 tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg
     6601 gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg
     6661 cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac
     6721 aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc
     6781 tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag
     6841 aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct
     6901 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     6961 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     7021 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     7081 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     7141 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     7201 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     7261 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     7321 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     7381 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     7441 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     7501 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     7561 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     7621 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cagagagggc
     7681 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     7741 agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     7801 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     7861 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     7921 accgaacccc taccaactgg tcggggtttg aaacggagac ggttgtacgt ctctttttta
     7981 agcttggcgt aactagatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa
     8041 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     8101 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     8161 ggacagcaga gatccacttt ggcgccggct cgagggggcc cgggtgcaaa gatggataaa
     8221 gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga aaggag
//
