LOCUS       sequence-198377-        7518 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Suppression of NFS1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7518)
  AUTHORS   Alvarez SW, Sviderskiy VO, Terzi EM, Papagiannakopoulos T, Moreira 
            AL, Adams S, Sabatini DM, Birsoy K, Possemato R
  TITLE     NFS1 undergoes positive selection in lung tumours and protects cells
            from ferroptosis.
  JOURNAL   Nature. 2017 Nov 22. doi: 10.1038/nature24637.
  PUBMED    29168506
REFERENCE   2  (bases 1 to 7518)
  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: "Nature. 
            2017 Nov 22. doi: 10.1038/nature24637."
COMMENT     Sequence Label: pLKO.1P shNFS1_2
FEATURES             Location/Qualifiers
     source          1..7518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    48..165
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        214..724
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     643..661
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             746..1345
                     /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(746..765)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1242..1262
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1361..1949
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1414..1434)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1832..1843)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2021..2254
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2332..2453
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2369..2388)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2423..2442
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2493..2628
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2648..2667)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2649..2667)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2677..2694)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2677..2693)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2686..2708)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2835..3290
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2922..2941)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3132..3153
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3316..3420
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3421..3489,3490..4281)
                     /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(3639..3658)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4452..5040
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4941..4960
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5194..5211
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5328..5349
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5364..5369,5370..5387,5388..5394)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5402..5418
                     /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     5407..5429
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5426..5442
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5426..5442
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5461..5481
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5463..5481
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5509..5735
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5736..5916
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5963..6088
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6581..6814
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6999..7043
                     /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        7221..7469
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7221..7241
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7392..7411
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 atttttgaat tctagatctt gagacaaatg gcagtattca tccacaattt taaaagaaaa
       61 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
      121 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
      181 gacagcagag atccactttg gcgccggctc gagggggccc gggttgcgcc ttttccaagg
      241 cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg
      301 cgccgaccct gggtctcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc
      361 cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc
      421 cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct
      481 cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa
      541 tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc
      601 ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa
      661 gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc
      721 ccagggggat ccaccggagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac
      781 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
      841 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
      901 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
      961 cgtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     1021 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     1081 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     1141 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     1201 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     1261 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     1321 catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc tggattacaa
     1381 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     1441 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     1501 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     1561 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     1621 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     1681 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     1741 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     1801 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     1861 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     1921 tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac ttacaaggca
     1981 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     2041 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
     2101 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     2161 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     2221 agaccctttt agtcagtgtg gaaaatctct agcagtacgt atagtagttc atgtcatctt
     2281 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt
     2341 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     2401 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     2461 tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     2521 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     2581 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc
     2641 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg
     2701 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     2761 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     2821 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2881 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2941 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3001 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3061 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3121 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3181 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3241 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     3301 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3361 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3421 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3481 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3541 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3601 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3661 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3721 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3781 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3841 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3901 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3961 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     4021 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4081 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4141 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4201 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4261 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4321 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4381 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4441 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4501 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4561 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     4621 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4681 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4741 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4801 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4861 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4921 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4981 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5041 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5101 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     5161 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     5221 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     5281 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     5341 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     5401 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
     5461 gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg
     5521 caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag
     5581 agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt gccttattag
     5641 gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag
     5701 atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct
     5761 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     5821 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     5881 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
     5941 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
     6001 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     6061 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     6121 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     6181 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     6241 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     6301 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     6361 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     6421 gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg
     6481 aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa
     6541 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt
     6601 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca
     6661 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc
     6721 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg
     6781 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac
     6841 tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga
     6901 tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa
     6961 tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg
     7021 aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata
     7081 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
     7141 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
     7201 caaccccgag gggacccaga gagggcctat ttcccatgat tccttcatat ttgcatatac
     7261 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
     7321 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     7381 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     7441 tatatatctt gtggaaagga cgaggtaccg gtgtgaagcg tcttcgagaa atctcgagat
     7501 ttctcgaaga cgcttcac
//
