LOCUS       sequence-213374-        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-sgULK1-1
FEATURES             Location/Qualifiers
     source          1..8893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1447..1680
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1865..1909
                     /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    2207..2324
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2585..2618)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2622..2642
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        2895..3106
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          3107..4045
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4002..4022
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4066..4662
                     /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(4066..4085)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4562..4582
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4672..4725
                     /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             4738..5436
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(5456..5489)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5545..6133
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5598..5618)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6016..6027)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6136..6152)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6137..6153)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6662..6842
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6904..7492)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6984..7003)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7663..8454,8455..8523))
                     /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     8286..8305
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8524..8628)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8703..8722)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac ctggaggagg
     1321 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     1381 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     1441 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     1501 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     1561 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     1621 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     1681 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     1741 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     1801 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     1861 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     1921 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     1981 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     2041 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     2101 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
     2161 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     2221 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2281 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2341 acagcagaga tccagtttgg ttagtaccgg gcccgccctg caggtctaga gatccgacgc
     2401 gccatctcta ggcccgcgcc ggccccctcg cacggacttg tgggagaagc tcggctactc
     2461 ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct taatgtgcga
     2521 taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat aggcttggat
     2581 ttctataact tcgtatagca tacattatac gaagttataa acagcacaaa aggaaactca
     2641 ccctaactgt aaagtaattg tgtgttttga gactataagt atcccttgga gaaccacctt
     2701 gttgggcggc ggcacagaga ccgtgtttaa gagctaagct ggaaacagca tagcaagttt
     2761 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttctc
     2821 gagtactagg atccattagg cggccgccct gcagggtgga taaccgtatt accgccatgc
     2881 atgtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     2941 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     3001 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     3061 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg
     3121 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc
     3181 cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag
     3241 ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg cctggcctgg
     3301 gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga
     3361 taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt tctggcaaga
     3421 tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg gggccgcggg
     3481 cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg
     3541 gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct
     3601 cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc
     3661 gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg
     3721 gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc
     3781 agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt
     3841 ctcgcgcttt tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt
     3901 tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc
     3961 cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt
     4021 tcaaagtttt tttcttccat ttcaggtgtc gtgagctagc ccaccatgac cgagtacaag
     4081 cccacggtgc gcctcgccac ccgcgacgac gtcccccggg ccgtacgcac cctcgccgcc
     4141 gcgttcgccg actaccccgc cacgcgccac accgtcgacc cggaccgcca catcgagcgg
     4201 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     4261 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     4321 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     4381 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     4441 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     4501 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     4561 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     4621 gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccggatcggg agagggcaga
     4681 ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaccggt cgccaccagc
     4741 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
     4801 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     4861 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     4921 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     4981 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     5041 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     5101 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     5161 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     5221 aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag atccaagaaa
     5281 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     5341 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     5401 gacctcccta gcaaactggg gcacaagctt aattaagaat tcgtcgaggg acctaataac
     5461 ttcgtatagc atacattata cgaagttata catgtttaag ggttccggtt ccactaggta
     5521 caattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     5581 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     5641 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     5701 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     5761 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     5821 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     5881 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     5941 tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     6001 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     6061 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     6121 gccgcctccc cgcatcgata ccgtcgacct cgatcgagac ctagaaaaac atggagcaat
     6181 cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga
     6241 ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc
     6301 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     6361 ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga
     6421 ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg gatggtgcta
     6481 caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag agaacacccg
     6541 cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tattagagtg
     6601 gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc cggactgtac
     6661 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     6721 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     6781 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     6841 cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     6901 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     6961 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     7021 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     7081 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     7141 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     7201 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     7261 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     7321 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     7381 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     7441 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     7501 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     7561 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     7621 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     7681 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7741 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     7801 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     7861 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     7921 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     7981 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     8041 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     8101 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     8161 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     8221 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     8281 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     8341 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     8401 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     8461 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     8521 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     8581 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     8641 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     8701 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     8761 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     8821 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     8881 agatatacgc gtt
//
