LOCUS       sequence-205395-        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     337..356
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     590..607
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    724..745
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(760..765,766..783,784..790)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    798..814
                     /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     803..825
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     822..838
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     822..838
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     857..877
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        859..877
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        905..1131
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1132..1312
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1359..1484
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1977..2210
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2395..2439
                     /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        2737..2985
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2737..2757
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2908..2927
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    3082..3199
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3248..3758
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3677..3695
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3780..4379
                     /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(3780..3799)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4276..4296
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4507..4740
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4812..4933
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4849..4868)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4903..4922
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4973..5108
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5128..5147)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5129..5147)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5157..5174)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5157..5173)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5166..5188)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5315..5770
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5402..5421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5612..5633
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5796..5900
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5901..5969,5970..6761)
                     /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(6119..6138)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6932..7084,1..436)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
       61 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
      121 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
      181 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
      241 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
      301 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
      361 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
      421 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
      481 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
      541 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
      601 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
      661 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
      721 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
      781 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      841 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
      901 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
      961 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
     1021 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
     1081 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
     1141 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     1201 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     1261 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
     1321 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
     1381 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
     1441 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
     1501 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
     1561 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
     1621 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
     1681 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
     1741 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
     1801 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
     1861 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1921 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1981 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     2041 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     2101 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     2161 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     2221 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     2281 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     2341 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     2401 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     2461 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     2521 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     2581 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa
     2641 ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg acggtatcga
     2701 tcacgagact agcctcgagc ggccgccccc ttcaccgagg gcctatttcc catgattcct
     2761 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     2821 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     2881 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     2941 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggcct aaggttaagt
     3001 cgccctcgct cgagcgaggg cgacttaacc ttaggttttt gaattctcga cctcgagaca
     3061 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     3121 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     3181 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac tttggccgcg
     3241 gctcgagggg gttggggttg cgccttttcc aaggcagccc tgggtttgcg cagggacgcg
     3301 gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct
     3361 tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga
     3421 cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt
     3481 gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc cagggagcaa
     3541 tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg
     3601 agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc
     3661 tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg
     3721 gctccctcgt tgaccgaatc accgacctct ctccccaggg ggatccaccg gagcttacca
     3781 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     3841 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     3901 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     3961 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     4021 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     4081 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     4141 ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca
     4201 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     4261 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     4321 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac
     4381 gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc acgaccccat gcatcggtac
     4441 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     4501 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg
     4561 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     4621 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     4681 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     4741 gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag
     4801 agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4861 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4921 tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc
     4981 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     5041 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     5101 tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     5161 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     5221 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     5281 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     5341 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     5401 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5461 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5521 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5581 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5641 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5701 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5761 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5821 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5881 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5941 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     6001 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     6061 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6121 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6181 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6241 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6301 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6361 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6421 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6481 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6541 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6601 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6661 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6721 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6781 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6841 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6901 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6961 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     7021 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     7081 ttct
//
