LOCUS       sequence-317443-        8363 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human non-targeting CRISPRi gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8363)
  AUTHORS   Lalli MA, Avey D, Dougherty JD, Milbrandt J, Mitra RD
  TITLE     High-throughput single-cell functional elucidation of 
            neurodevelopmental disease-associated genes reveals convergent 
            mechanisms altering neuronal differentiation.
  JOURNAL   Genome Res. 2020 Sep;30(9):1317-1331. doi: 10.1101/gr.262295.120. 
            Epub 2020 Sep 4.
  PUBMED    32887689
REFERENCE   2  (bases 1 to 8363)
  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"; doi: 
            "10.1101/gr.262295.120"; journalName: "Genome Res"; date: "2020-09";
            volume: "30"; issue: "9"; pages: "1317-1331"
COMMENT     Sequence Label: Non-targeting CRISPRi plasmid
FEATURES             Location/Qualifiers
     source          1..8363
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      390..978
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     879..898
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1132..1149
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1266..1287
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1302..1307,1308..1325,1326..1332)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1340..1356
                     /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     1345..1367
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1364..1380
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1364..1380
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1399..1419
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1401..1419
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1447..1673
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1674..1854
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1901..2026
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2519..2752
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2937..2981
                     /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    3162..3279
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3418..4596
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3649..4587
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4544..4564
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4609..5208
                     /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(4609..4628)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5105..5125
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5224..5812
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5277..5297)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5695..5706)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        5968..6216
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5968..5988
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6139..6158
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     LTR             6375..6555
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    6633..6754
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6670..6689)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6724..6743
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6794..6929
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6949..6968)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6950..6968)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6978..6995)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6978..6994)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6987..7009)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7136..7591
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7223..7242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7433..7454
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7617..7721
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7722..7790,7791..8363,1..219)
                     /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(7940..7959)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
       61 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
      121 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
      181 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
      241 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
      301 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
      361 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
      421 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
      481 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
      541 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
      601 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
      661 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
      721 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
      781 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
      841 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
      901 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
      961 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     1021 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     1081 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     1141 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     1201 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     1261 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     1321 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     1381 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa
     1441 gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc
     1501 aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt
     1561 acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg
     1621 aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc
     1681 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     1741 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     1801 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     1861 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     1921 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     1981 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     2041 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     2101 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     2161 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     2221 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     2281 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     2341 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga
     2401 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag
     2461 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag
     2521 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga
     2581 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc
     2641 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc
     2701 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt
     2761 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta
     2821 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta
     2881 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga
     2941 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa
     3001 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
     3061 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
     3121 cgtttcagaa tcttgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg
     3181 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact
     3241 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc
     3301 agagatccac tttggcgccg gctcgagggg gcccgggtgc aaagatggat aaagttttaa
     3361 acagagagga atctttgcag ctaatggacc ttctaggtct tgaaaggagt gggaattggc
     3421 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     3481 ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     3541 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     3601 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg
     3661 tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact
     3721 tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag tgggtgggag
     3781 agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga ggcctggcct
     3841 gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc
     3901 gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa
     3961 gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt tggggccgcg
     4021 ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg
     4081 cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct ggtgcctggc
     4141 ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt
     4201 gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa atggaggacg
     4261 cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc
     4321 tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca cctcgattag
     4381 ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta tgcgatggag
     4441 tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt gatgtaattc
     4501 tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc tcagacagtg
     4561 gttcaaagtt tttttcttcc atttcaggtg tcgtgacgta cggccaccat gaccgagtac
     4621 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     4681 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     4741 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     4801 tgggtcgcgg acgacggcgc cgccgtggcg gtctggacca cgccggagag cgtcgaagcg
     4861 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     4921 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     4981 ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     5041 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     5101 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     5161 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc
     5221 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     5281 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     5341 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     5401 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     5461 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     5521 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     5581 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     5641 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     5701 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     5761 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
     5821 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     5881 gactggaagg gctaattcac tcccaacgaa gacaagattt caccattatc gtttcagacc
     5941 cacctcccaa ccccgagggg acccagagag ggcctatttc ccatgattcc ttcatatttg
     6001 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     6061 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     6121 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     6181 ttggctttat atatcttgtg gaaaggacga aacaccggag gcgaggtaag acgcgggttt
     6241 aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga
     6301 aaaagtggca ccgagtcggt gcttttttaa gcttggcgta actagatctt gagacactgc
     6361 tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
     6421 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     6481 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     6541 ggaaaatctc tagcagtacg tatagtagtt catgtcatct tattattcag tatttataac
     6601 ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt
     6661 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     6721 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc
     6781 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     6841 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     6901 gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag
     6961 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     7021 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     7081 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     7141 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     7201 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     7261 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     7321 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     7381 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     7441 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     7501 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     7561 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     7621 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     7681 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     7741 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     7801 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     7861 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     7921 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     7981 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     8041 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     8101 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     8161 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     8221 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     8281 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     8341 actggatgga ggcggataaa gtt
//
