LOCUS       sequence-421504-        8106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6-sgRNA-EF1a-mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8106)
  AUTHORS   Tan IL, Perez AR, Lew RJ, Sun X, Baldwin A, Zhu YK, Shah MM, Berger 
            MS, Doudna JA, Fellmann C
  TITLE     Targeting the non-coding genome and temozolomide signature enables 
            CRISPR-mediated glioma oncolysis.
  JOURNAL   Cell Rep. 2023 Nov 28;42(11):113339. doi: 
            10.1016/j.celrep.2023.113339. Epub 2023 Nov 2.
  PUBMED    37917583
REFERENCE   2  (bases 1 to 8106)
  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: "Cell Rep.";
            date: "2023-11-28"; volume: "42(11):113339. doi"; pages: " 
            10.1016/j.celrep.2023.113339. Epub 2023 Nov 2"
COMMENT     Sequence Label: pCF821-sgNT-3_U6-sgRNA-EF1a-mNeonGreen
FEATURES             Location/Qualifiers
     source          1..8106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        232..611
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        613..811
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     766..786
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             829..1009
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1056..1181
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1674..1907
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2092..2136
                     /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"
     misc_feature    2434..2551
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2602..2850
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2602..2622
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2773..2792
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3001..4178
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3232..4169
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4126..4146
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4196..4205
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    4928..5516
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4981..5001)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5399..5410)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5588..5821
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5847..5864)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5853..6077
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6183..6200)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6354..6942)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6434..6453)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7113..7904,7905..7973))
                     /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     7736..7755
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7974..8078)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat tgacattgat
      241 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      301 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      361 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      421 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      481 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      541 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      601 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      661 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      721 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      781 ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg
      841 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      901 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      961 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
     1021 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
     1081 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
     1141 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
     1201 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
     1261 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
     1321 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
     1381 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1441 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1501 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1561 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1621 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1681 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1741 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1801 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1861 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1921 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1981 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     2041 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     2101 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     2161 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     2221 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     2281 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     2341 ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact gcgtgcgcca
     2401 attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     2461 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     2521 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     2581 agtttggtta attaaggtac cgagggccta tttcccatga ttccttcata tttgcatata
     2641 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     2701 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2761 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2821 ttatatatct tgtggaaagg acgaaacacc ggacgactag ttaggcgtgt agtttaagag
     2881 ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
     2941 tggcaccgag tcggtgcttt ttttgaattc gctagctagg tcttgaaagg agtgggaatt
     3001 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     3061 ggaggggtcg gcaattgatc cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     3121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     3181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
     3241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
     3301 acttccactg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     3361 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     3421 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     3481 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     3541 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     3601 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     3661 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     3721 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     3781 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     3841 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     3901 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     3961 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     4021 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     4081 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     4141 tggttcaaag tttttttctt ccatttcagg tgtcgtgaac cggttctaga gcgctgccac
     4201 catggtcagt aagggcgaag aagataatat ggcttccttg cctgccacac atgaactcca
     4261 catattcggg tctatcaacg gcgtggactt cgacatggtt gggcaaggaa ccggaaatcc
     4321 caacgatgga tacgaggagc tgaacctgaa gtcaacgaaa ggtgatcttc aattctcccc
     4381 ctggattctc gtccctcaca ttggctatgg tttccaccag tacctcccgt accctgacgg
     4441 gatgtctcca ttccaagccg caatggtaga tgggtcaggc taccaagttc acagaactat
     4501 gcagtttgaa gatggtgcat ctctgacagt aaattacagg tatacctacg aaggttcaca
     4561 catcaaagga gaagctcagg tgaaaggtac gggattccca gcagacggcc cagtaatgac
     4621 caattccctc acggctgccg actggtgcag gagtaaaaag acatatccaa atgacaagac
     4681 tattatctct actttcaaat ggtcttatac gacaggcaac ggaaagaggt atcggtcaac
     4741 cgcgcgcaca acatatacgt ttgctaagcc tatggctgcc aattatctga aaaatcagcc
     4801 catgtatgta tttagaaaaa ccgaactgaa gcactccaaa actgaattga acttcaaaga
     4861 gtggcaaaag gccttcaccg atgtcatggg catggatgaa ttgtataagt gaacgcgtta
     4921 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     4981 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     5041 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     5101 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     5161 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     5221 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     5281 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     5341 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     5401 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     5461 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg
     5521 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     5581 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
     5641 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     5701 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     5761 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     5821 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     5881 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     5941 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     6001 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     6061 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc cattaatgaa aagaaccagc
     6121 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     6181 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     6241 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     6301 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     6361 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     6421 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6481 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6541 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6601 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6661 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6721 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6781 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6841 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6901 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6961 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     7021 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     7081 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     7141 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     7201 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     7261 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     7321 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7381 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7441 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7501 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7561 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7621 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7681 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7741 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7801 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7861 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7921 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7981 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     8041 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     8101 cctgac
//
