LOCUS       sequence-431901-        7645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CRISPRi gRNA for CD3e promoter and GFP. gRNA driven by 
            mU6..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7645)
  AUTHORS   Pacalin NM, Steinhart Z, Shi Q, Belk JA, Dorovskyi D, Kraft K, 
            Parker KR, Shy BR, Marson A, Chang HY
  TITLE     Bidirectional epigenetic editing reveals hierarchies in gene 
            regulation.
  JOURNAL   Nat Biotechnol. 2024 May 17. doi: 10.1038/s41587-024-02213-3.
  PUBMED    38760566
REFERENCE   2  (bases 1 to 7645)
  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: "Nat 
            Biotechnol. 2024 May 17. doi: 10.1038/s41587-024-02213-3."
COMMENT     Sequence Label: p104_gRNA_Sp_CD3e
FEATURES             Location/Qualifiers
     source          1..7645
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        183..486
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        487..690
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             join(724..726,727..729,730..1443)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(769..790)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1030..1049)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1377..1398
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1463..1496)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1552..2140
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1605..1625)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2023..2034)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2143..2159)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2144..2160)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             2669..2849
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(2911..3499)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2991..3010)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3670..4461,4462..4530))
                     /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     4293..4312
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4531..4635)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4710..4729)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4901..5280
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5282..5480
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             5498..5678
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5725..5850
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6347..6580
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6765..6809
                     /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    7107..7224
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(7477..7510)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     7514..7534
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
ORIGIN
        1 gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
       61 tttttctcga gtactaggat ccattaggcg gccgcgtgga taaccgtatt accgccatgc
      121 attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      181 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      241 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      301 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      361 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      421 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      481 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      541 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      601 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      661 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagccc
      721 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
      781 gacggcgacg taaacggcca caagttcagc gtgtctggcg agggcgaggg cgatgccacc
      841 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
      901 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
      961 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1021 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1081 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1141 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     1201 aacggcatca aggcgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     1261 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1321 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     1381 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     1441 taagaattcg tcgagggacc taataacttc gtatagcata cattatacga agttatacat
     1501 gtttaagggt tccggttcca ctaggtacaa ttcgatatca agcttatcga taatcaacct
     1561 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     1621 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     1681 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     1741 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     1801 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     1861 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     1921 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     1981 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     2041 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     2101 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga
     2161 tcgagaccta gaaaaacatg gagcaatcac aagtagcaat acagcagcta ccaatgctga
     2221 ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt tttccagtca cacctcaggt
     2281 acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
     2341 ggggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat
     2401 ctaccacaca caaggctact tccctgattg gcagaactac acaccagggc cagggatcag
     2461 atatccactg acctttggat ggtgctacaa gctagtacca gttgagcaag agaaggtaga
     2521 agaagccaat gaaggagaga acacccgctt gttacaccct gtgagcctgc atgggatgga
     2581 tgacccggag agagaagtat tagagtggag gtttgacagc cgcctagcat ttcatcacat
     2641 ggcccgagag ctgcatccgg actgtactgg gtctctctgg ttagaccaga tctgagcctg
     2701 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
     2761 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
     2821 cttttagtca gtgtggaaaa tctctagcag catgtgagca aaaggccagc aaaaggccag
     2881 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2941 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3001 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3061 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3121 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3181 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3241 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3301 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3361 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3421 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3481 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3541 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3601 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3661 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3721 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3781 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     3841 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3901 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3961 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4021 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4081 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4141 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4201 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4261 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4321 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4381 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4441 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4501 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4561 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4621 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     4681 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4741 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     4801 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
     4861 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
     4921 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     4981 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     5041 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     5101 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     5161 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     5221 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     5281 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     5341 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     5401 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     5461 tgggaggtct atataagcag cgcgttttgc ctgtactggg tctctctggt tagaccagat
     5521 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     5581 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     5641 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg
     5701 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc
     5761 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc
     5821 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga
     5881 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta
     5941 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa
     6001 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt
     6061 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa
     6121 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca
     6181 cagcaagcgg ccggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     6241 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     6301 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     6361 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     6421 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     6481 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     6541 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     6601 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     6661 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     6721 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     6781 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     6841 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     6901 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     6961 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     7021 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc
     7081 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     7141 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     7201 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg
     7261 ttagtaccgg gcccgctcta gagatccgac gcgccatctc taggcccgcg ccggccccct
     7321 cgcacggact tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata
     7381 tttcctagta actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa
     7441 tactagctac attttacatg ataggcttgg atttctataa cttcgtatag catacattat
     7501 acgaagttat aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt
     7561 gagactataa gtatcccttg gagaaccacc ttgttggcca agtgaggtaa aacccgcgtt
     7621 taagagctat gctggaaaca gcata
//
