LOCUS       sequence-213418-        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-shFAM134B
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      135..723
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     624..643
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     877..894
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1011..1032
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1047..1052,1053..1070,1071..1077)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1085..1101
                     /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     1090..1112
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1109..1125
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1109..1125
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1144..1164
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1146..1164
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1192..1418
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1419..1599
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1646..1771
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2264..2497
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2682..2726
                     /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        3024..3272
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3024..3044
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3195..3214
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    3369..3486
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3535..4045
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3964..3982
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4067..4666
                     /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(4067..4086)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4563..4583
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4794..5027
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5099..5220
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5136..5155)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5190..5209
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5260..5395
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5415..5434)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5416..5434)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5444..5461)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5444..5460)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5453..5475)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5602..6057
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5689..5708)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5899..5920
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6083..6187
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6188..6256,6257..7048)
                     /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(6406..6425)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
       61 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
      121 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
      181 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
      241 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
      301 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
      361 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
      421 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
      481 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
      541 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
      601 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
      661 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
      721 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
      781 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
      841 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      901 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
      961 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     1021 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     1081 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     1141 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt
     1201 atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag
     1261 gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat
     1321 taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca
     1381 gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga
     1441 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     1501 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     1561 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
     1621 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
     1681 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
     1741 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
     1801 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     1861 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     1921 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     1981 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     2041 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     2101 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     2161 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     2221 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     2281 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     2341 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     2401 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     2461 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     2521 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     2581 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     2641 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     2701 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     2761 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     2821 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
     2881 tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag
     2941 acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgatca cgagactagc
     3001 ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata
     3061 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
     3121 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     3181 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     3241 ctttatatat cttgtggaaa ggacgaaaca ccgggaggta tcctggactg ataatctcga
     3301 gattatcagt ccaggatacc tctttttgaa ttctcgacct cgagacaaat ggcagtattc
     3361 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     3421 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     3481 aattttcggg tttattacag ggacagcaga gatccacttt ggccgcggct cgagggggtt
     3541 ggggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct gctctgggcg
     3601 tggttccggg aaacgcagcg gcgccgaccc tgggtctcgc acattcttca cgtccgttcg
     3661 cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc
     3721 gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc
     3781 cgcacgtctc actagtaccc tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg
     3841 accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga gcagcggccg
     3901 ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc
     3961 gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct ccctcgttga
     4021 ccgaatcacc gacctctctc cccaggggga tccaccggag cttaccatga ccgagtacaa
     4081 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc
     4141 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg
     4201 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     4261 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     4321 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     4381 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     4441 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     4501 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
     4561 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     4621 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc cgccccacga
     4681 cccgcagcgc ccgaccgaaa ggagcgcacg accccatgca tcggtacctt taagaccaat
     4741 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
     4801 gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt
     4861 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     4921 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     4981 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg
     5041 tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa
     5101 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     5161 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5221 tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc
     5281 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     5341 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg
     5401 acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
     5461 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     5521 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     5581 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     5641 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     5701 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     5761 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     5821 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5881 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     5941 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     6001 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     6061 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     6121 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     6181 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     6241 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     6301 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     6361 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     6421 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     6481 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     6541 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     6601 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     6661 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     6721 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     6781 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6841 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6901 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6961 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     7021 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     7081 gatt
//
