LOCUS       sequence-213363-        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-sgATL2-2
FEATURES             Location/Qualifiers
     source          1..8893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..212
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          213..1151
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1108..1128
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1172..1768
                     /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(1172..1191)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1668..1688
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1778..1831
                     /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             1844..2542
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(2562..2595)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2651..3239
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2704..2724)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3122..3133)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3242..3258)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3243..3259)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3768..3948
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4010..4598)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4090..4109)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4769..5560,5561..5629))
                     /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     5392..5411
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5630..5734)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5809..5828)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6000..6379
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6381..6579
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6534..6554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6597..6777
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6824..6949
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7446..7679
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7864..7908
                     /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    8206..8323
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(8584..8617)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     8621..8641
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
ORIGIN
        1 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
       61 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      121 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      181 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
      241 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
      301 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
      361 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
      421 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
      481 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
      541 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
      601 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
      661 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
      721 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
      781 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc
      841 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
      901 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg cgcttttgga
      961 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
     1021 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
     1081 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
     1141 ttccatttca ggtgtcgtga gctagcccac catgaccgag tacaagccca cggtgcgcct
     1201 cgccacccgc gacgacgtcc cccgggccgt acgcaccctc gccgccgcgt tcgccgacta
     1261 ccccgccacg cgccacaccg tcgacccgga ccgccacatc gagcgggtca ccgagctgca
     1321 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     1381 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     1441 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     1501 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     1561 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     1621 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     1681 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     1741 ctggtgcatg acccgcaagc ccggtgccgg atcgggagag ggcagaggaa gtctgctaac
     1801 atgcggtgac gtcgaggaga atcctggccc accggtcgcc accagcgagc tgattaagga
     1861 gaacatgcac atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac
     1921 atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga
     1981 gggcggccct ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa
     2041 gaccttcatc aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg
     2101 cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga
     2161 caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac
     2221 atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct
     2281 gtaccccgct gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg
     2341 gagccatctg atcgcaaaca tcaagaccac atatagatcc aagaaacccg ctaagaacct
     2401 caagatgcct ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa
     2461 cgagacctac gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa
     2521 actggggcac aagcttaatt aagaattcgt cgagggacct aataacttcg tatagcatac
     2581 attatacgaa gttatacatg tttaagggtt ccggttccac taggtacaat tcgatatcaa
     2641 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     2701 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     2761 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     2821 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     2881 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     2941 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3001 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3061 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     3121 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     3181 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     3241 tcgataccgt cgacctcgat cgagacctag aaaaacatgg agcaatcaca agtagcaata
     3301 cagcagctac caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt
     3361 ttccagtcac acctcaggta cctttaagac caatgactta caaggcagct gtagatctta
     3421 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     3481 atatccttga tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca
     3541 caccagggcc agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag
     3601 ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg
     3661 tgagcctgca tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc
     3721 gcctagcatt tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt
     3781 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     3841 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     3901 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa
     3961 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4021 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4081 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4141 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4201 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4261 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4321 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4381 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4441 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4501 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4561 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4621 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4681 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4741 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4801 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4861 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4921 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4981 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5041 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5101 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5161 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5221 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5281 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5341 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5401 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5461 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5521 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5581 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5641 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5701 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5761 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
     5821 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     5881 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     5941 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
     6001 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     6061 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     6121 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     6181 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     6241 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     6301 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     6361 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     6421 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     6481 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     6541 gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt
     6601 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     6661 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     6721 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg
     6781 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac
     6841 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa
     6901 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg
     6961 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata
     7021 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg
     7081 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca
     7141 gacaggatca gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca
     7201 tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa
     7261 caaaagtaag accaccgcac agcaagcggc cggccgctga tcttcagacc tggaggagga
     7321 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     7381 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     7441 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     7501 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     7561 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     7621 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     7681 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     7741 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     7801 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     7861 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     7921 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     7981 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     8041 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     8101 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     8161 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     8221 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     8281 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     8341 cagcagagat ccagtttggt tagtaccggg cccgccctgc aggtctagag atccgacgcg
     8401 ccatctctag gcccgcgccg gccccctcgc acggacttgt gggagaagct cggctactcc
     8461 cctgccccgg ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat
     8521 aaaagacaga taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt
     8581 tctataactt cgtatagcat acattatacg aagttataaa cagcacaaaa ggaaactcac
     8641 cctaactgta aagtaattgt gtgttttgag actataagta tcccttggag aaccaccttg
     8701 ttggtagctg ctgggagaac caggtttaag agctaagctg gaaacagcat agcaagttta
     8761 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttctcg
     8821 agtactagga tccattaggc ggccgccctg cagggtggat aaccgtatta ccgccatgca
     8881 tgtgcccgtc agt
//
