LOCUS       sequence-213416-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  short-hairpin knockdown of targeted gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  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 7084)
  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: pLKO.1-puro-shNT
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    288..309
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(324..329,330..347,348..354)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    362..378
                     /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     367..389
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     386..402
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     386..402
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     421..441
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        423..441
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        469..695
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             696..876
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    923..1048
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1541..1774
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1959..2003
                     /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"
     promoter        2301..2549
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2301..2321
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2472..2491
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2646..2763
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2812..3322
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3241..3259
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3344..3943
                     /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(3344..3363)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3840..3860
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4071..4304
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4376..4497
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4413..4432)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4467..4486
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4537..4672
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4692..4711)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4693..4711)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4721..4738)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4721..4737)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4730..4752)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4879..5334
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4966..4985)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5176..5197
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5360..5464
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5465..5533,5534..6325)
                     /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(5683..5702)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6496..7084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6985..7004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      121 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      181 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
      241 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
      301 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
      361 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
      421 gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg
      481 caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag
      541 agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt gccttattag
      601 gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag
      661 atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct
      721 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
      781 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
      841 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
      901 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
      961 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     1021 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     1081 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     1141 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     1201 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     1261 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     1321 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     1381 gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg
     1441 aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa
     1501 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt
     1561 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca
     1621 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc
     1681 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg
     1741 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac
     1801 tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga
     1861 tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa
     1921 tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg
     1981 aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata
     2041 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
     2101 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
     2161 caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca
     2221 gagacagatc cattcgatta gtgaacggat ctcgacggta tcgatcacga gactagcctc
     2281 gagcggccgc ccccttcacc gagggcctat ttcccatgat tccttcatat ttgcatatac
     2341 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     2401 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     2461 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     2521 tatatatctt gtggaaagga cgaaacaccg gcctaaggtt aagtcgccct cgctcgagcg
     2581 agggcgactt aaccttaggt ttttgaattc tcgacctcga gacaaatggc agtattcatc
     2641 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     2701 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     2761 tttcgggttt attacaggga cagcagagat ccactttggc cgcggctcga gggggttggg
     2821 gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg
     2881 ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag
     2941 cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc
     3001 cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc
     3061 acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc
     3121 gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga
     3181 aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg
     3241 gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg
     3301 aatcaccgac ctctctcccc agggggatcc accggagctt accatgaccg agtacaagcc
     3361 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     3421 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     3481 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     3541 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     3601 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     3661 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     3721 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     3781 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     3841 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     3901 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc
     3961 gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg gtacctttaa gaccaatgac
     4021 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     4081 aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga
     4141 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     4201 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     4261 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca
     4321 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
     4381 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4441 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4501 tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4561 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4621 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg
     4681 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     4741 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     4801 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     4861 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4921 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4981 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5041 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5101 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5161 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5221 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5281 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     5341 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5401 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5461 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5521 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5581 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5641 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5701 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5761 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5821 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5881 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5941 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6001 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6061 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6121 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6181 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6241 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6301 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6361 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6421 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6481 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6541 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6601 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6661 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6721 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6781 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6841 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6901 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6961 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     7021 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     7081 gaaa
//
