LOCUS       sequence-215170-        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-sgATL1-5
FEATURES             Location/Qualifiers
     source          1..8893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             278..458
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(520..1108)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(600..619)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1279..2070,2071..2139))
                     /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     1902..1921
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2140..2244)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2319..2338)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2510..2889
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2891..3089
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3044..3064
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             3107..3287
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3334..3459
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3956..4189
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4374..4418
                     /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    4716..4833
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(5094..5127)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     5131..5151
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        5404..5615
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          5616..6554
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     6511..6531
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             6575..7171
                     /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(6575..6594)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7071..7091
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             7181..7234
                     /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             7247..7945
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(7965..7998)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    8054..8642
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8107..8127)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8525..8536)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8645..8661)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8646..8662)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca caccagggcc
       61 agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag ttgagcaaga
      121 gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg tgagcctgca
      181 tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc gcctagcatt
      241 tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt tagaccagat
      301 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
      361 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
      421 cctcagaccc ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa aaggccagca
      481 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      541 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      601 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
      661 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
      721 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
      781 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
      841 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
      901 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
      961 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     1021 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     1081 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     1141 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     1201 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1261 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     1321 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     1381 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     1441 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     1501 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     1561 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     1621 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     1681 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     1741 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     1801 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     1861 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     1921 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     1981 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     2041 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     2101 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     2161 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2221 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     2281 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
     2341 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
     2401 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
     2461 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
     2521 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     2581 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     2641 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     2701 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     2761 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     2821 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     2881 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
     2941 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     3001 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
     3061 cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt
     3121 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     3181 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     3241 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
     3301 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
     3361 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
     3421 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
     3481 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
     3541 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
     3601 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
     3661 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
     3721 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
     3781 accaccgcac agcaagcggc cggccgctga tcttcagacc tggaggagga gatatgaggg
     3841 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     3901 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     3961 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     4021 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     4081 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     4141 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     4201 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     4261 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     4321 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     4381 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     4441 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     4501 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     4561 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
     4621 gtggagagag agacagagac agatccattc gattagtgaa cggatcggca ctgcgtgcgc
     4681 caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
     4741 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     4801 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
     4861 ccagtttggt tagtaccggg cccgccctgc aggtctagag atccgacgcg ccatctctag
     4921 gcccgcgccg gccccctcgc acggacttgt gggagaagct cggctactcc cctgccccgg
     4981 ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat aaaagacaga
     5041 taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt tctataactt
     5101 cgtatagcat acattatacg aagttataaa cagcacaaaa ggaaactcac cctaactgta
     5161 aagtaattgt gtgttttgag actataagta tcccttggag aaccaccttg ttggtcctcc
     5221 caaccgatcg ctagtttaag agctaagctg gaaacagcat agcaagttta aataaggcta
     5281 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttctcg agtactagga
     5341 tccattaggc ggccgccctg cagggtggat aaccgtatta ccgccatgca tgtgcccgtc
     5401 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     5461 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     5521 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     5581 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg
     5641 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc acctggctgc
     5701 agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt
     5761 gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc
     5821 gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag
     5881 ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa
     5941 atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg
     6001 cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa
     6061 tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt
     6121 gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa
     6181 gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag
     6241 agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt
     6301 catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgcgctttt
     6361 ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg
     6421 agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg
     6481 ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt
     6541 ttcttccatt tcaggtgtcg tgagctagcc caccatgacc gagtacaagc ccacggtgcg
     6601 cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg cgttcgccga
     6661 ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg tcaccgagct
     6721 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     6781 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     6841 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     6901 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     6961 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     7021 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     7081 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     7141 cacctggtgc atgacccgca agcccggtgc cggatcggga gagggcagag gaagtctgct
     7201 aacatgcggt gacgtcgagg agaatcctgg cccaccggtc gccaccagcg agctgattaa
     7261 ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg
     7321 cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt
     7381 cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag
     7441 caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga
     7501 gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca
     7561 ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt
     7621 cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgagac
     7681 gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg
     7741 cgggagccat ctgatcgcaa acatcaagac cacatataga tccaagaaac ccgctaagaa
     7801 cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa
     7861 caacgagacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag
     7921 caaactgggg cacaagctta attaagaatt cgtcgaggga cctaataact tcgtatagca
     7981 tacattatac gaagttatac atgtttaagg gttccggttc cactaggtac aattcgatat
     8041 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     8101 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     8161 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     8221 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     8281 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     8341 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     8401 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     8461 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     8521 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     8581 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     8641 gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc acaagtagca
     8701 atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg
     8761 gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     8821 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     8881 aagatatcct tga
//
