LOCUS       sequence-213376-        8893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRi knockdown of targeted gene (to be used with Addgene #102244 
            or other dCas9-KRAB constructs).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8893)
  AUTHORS   Liang JR, Lingeman E, Ahmed S, Corn JE
  TITLE     Atlastins remodel the endoplasmic reticulum for selective autophagy.
  JOURNAL   J Cell Biol. 2018 Aug 24. pii: jcb.201804185. doi: 
            10.1083/jcb.201804185.
  PUBMED    30143524
REFERENCE   2  (bases 1 to 8893)
  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: "J Cell 
            Biol. 2018 Aug 24. pii: jcb.201804185. doi: 10.1083/jcb.201804185."
COMMENT     Sequence Label: pLG1-puro Non-targeting sgRNA 3
FEATURES             Location/Qualifiers
     source          1..8893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..200
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             218..398
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    445..570
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1067..1300
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1485..1529
                     /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    1827..1944
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2205..2238)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2242..2262
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        2515..2726
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          2727..3665
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3622..3642
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3686..4282
                     /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(3686..3705)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4182..4202
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4292..4345
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4358..5056
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(5076..5109)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5165..5753
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5218..5238)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5636..5647)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5756..5772)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5757..5773)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6282..6462
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6524..7112)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6604..6623)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7283..8074,8075..8143))
                     /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     7906..7925
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8144..8248)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8323..8342)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        8514..8893
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag cgcgttttgc ctgtactggg tctctctggt tagaccagat
      241 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
      301 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
      361 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg
      421 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc
      481 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc
      541 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga
      601 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta
      661 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa
      721 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt
      781 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa
      841 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca
      901 cagcaagcgg ccggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
      961 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     1021 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     1081 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     1141 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     1201 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     1261 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     1321 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     1381 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     1441 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     1501 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     1561 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     1621 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     1681 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     1741 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc
     1801 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     1861 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     1921 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg
     1981 ttagtaccgg gcccgccctg caggtctaga gatccgacgc gccatctcta ggcccgcgcc
     2041 ggccccctcg cacggacttg tgggagaagc tcggctactc ccctgccccg gttaatttgc
     2101 atataatatt tcctagtaac tatagaggct taatgtgcga taaaagacag ataatctgtt
     2161 ctttttaata ctagctacat tttacatgat aggcttggat ttctataact tcgtatagca
     2221 tacattatac gaagttataa acagcacaaa aggaaactca ccctaactgt aaagtaattg
     2281 tgtgttttga gactataagt atcccttgga gaaccacctt gttgggctcg gtcccgcgtc
     2341 gtcggtttaa gagctaagct ggaaacagca tagcaagttt aaataaggct agtccgttat
     2401 caacttgaaa aagtggcacc gagtcggtgc tttttttctc gagtactagg atccattagg
     2461 cggccgccct gcagggtgga taaccgtatt accgccatgc atgtgcccgt cagtgggcag
     2521 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg
     2581 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
     2641 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
     2701 gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc
     2761 tctttacggg ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga
     2821 ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag
     2881 gagccccttc gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc
     2941 gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa
     3001 atttttgatg acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc
     3061 aagatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt
     3121 cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg
     3181 ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc
     3241 cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc
     3301 ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg
     3361 gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact
     3421 ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgcgcttt tggagtacgt
     3481 cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg
     3541 agactgaagt taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg
     3601 agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat
     3661 ttcaggtgtc gtgagctagc ccaccatgac cgagtacaag cccacggtgc gcctcgccac
     3721 ccgcgacgac gtcccccggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     3781 cacgcgccac accgtcgacc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     3841 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     3901 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     3961 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     4021 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     4081 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     4141 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     4201 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     4261 catgacccgc aagcccggtg ccggatcggg agagggcaga ggaagtctgc taacatgcgg
     4321 tgacgtcgag gagaatcctg gcccaccggt cgccaccagc gagctgatta aggagaacat
     4381 gcacatgaag ctgtacatgg agggcaccgt ggacaaccat cacttcaagt gcacatccga
     4441 gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg
     4501 ccctctcccc ttcgccttcg acatcctggc tactagcttc ctctacggca gcaagacctt
     4561 catcaaccac acccagggca tccccgactt cttcaagcag tccttccctg agggcttcac
     4621 atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag
     4681 cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcacatccaa
     4741 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc
     4801 cgctgacggc ggcctggaag gcagaaacga catggccctg aagctcgtgg gcgggagcca
     4861 tctgatcgca aacatcaaga ccacatatag atccaagaaa cccgctaaga acctcaagat
     4921 gcctggcgtc tactatgtgg actacagact ggaaagaatc aaggaggcca acaacgagac
     4981 ctacgtcgag cagcacgagg tggcagtggc cagatactgc gacctcccta gcaaactggg
     5041 gcacaagctt aattaagaat tcgtcgaggg acctaataac ttcgtatagc atacattata
     5101 cgaagttata catgtttaag ggttccggtt ccactaggta caattcgata tcaagcttat
     5161 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     5221 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     5281 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     5341 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     5401 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     5461 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     5521 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     5581 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     5641 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     5701 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata
     5761 ccgtcgacct cgatcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag
     5821 ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag
     5881 tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact
     5941 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc
     6001 ttgatctgtg gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag
     6061 ggccagggat cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc
     6121 aagagaaggt agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc
     6181 tgcatgggat ggatgacccg gagagagaag tattagagtg gaggtttgac agccgcctag
     6241 catttcatca catggcccga gagctgcatc cggactgtac tgggtctctc tggttagacc
     6301 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     6361 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     6421 gatccctcag acccttttag tcagtgtgga aaatctctag cagcatgtga gcaaaaggcc
     6481 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6541 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6601 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6661 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6721 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6781 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6841 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6901 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6961 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     7021 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     7081 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     7141 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     7201 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     7261 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     7321 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7381 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7441 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7501 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7561 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7621 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7681 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7741 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7801 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7861 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7921 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7981 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     8041 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     8101 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     8161 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     8221 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     8281 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     8341 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     8401 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     8461 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
     8521 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     8581 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     8641 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     8701 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     8761 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     8821 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     8881 cgctattacc atg
//
