LOCUS       sequence-198378-        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_3
FEATURES             Location/Qualifiers
     source          1..7518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    40..157
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        206..716
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     635..653
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             738..1337
                     /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(738..757)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1234..1254
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1353..1941
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1406..1426)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1824..1835)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2013..2246
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2324..2445
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2361..2380)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2415..2434
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2485..2620
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2640..2659)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2641..2659)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2669..2686)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2669..2685)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2678..2700)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2827..3282
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2914..2933)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3124..3145
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3308..3412
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3413..3481,3482..4273)
                     /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(3631..3650)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4444..5032
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4933..4952
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5186..5203
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5320..5341
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5356..5361,5362..5379,5380..5386)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5394..5410
                     /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     5399..5421
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5418..5434
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5418..5434
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5453..5473
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5455..5473
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5501..5727
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5728..5908
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5955..6080
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6573..6806
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6991..7035
                     /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        7213..7461
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7213..7233
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7384..7403
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 attctagatc ttgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
       61 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
      121 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
      181 agatccactt tggcgccggc tcgagggggc ccgggttgcg ccttttccaa ggcagccctg
      241 ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc
      301 ctgggtctcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc
      361 ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt
      421 tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc ctcgcagacg
      481 gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct
      541 gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg
      601 gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg
      661 agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct ccccaggggg
      721 atccaccgga gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
      781 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
      841 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
      901 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gccgtggcgg
      961 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     1021 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     1081 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     1141 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     1201 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     1261 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     1321 gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg
     1381 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     1441 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     1501 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     1561 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     1621 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     1681 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     1741 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     1801 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     1861 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     1921 ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga
     1981 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     2041 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     2101 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     2161 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     2221 ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca
     2281 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc
     2341 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     2401 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta
     2461 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2521 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     2581 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc
     2641 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     2701 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     2761 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     2821 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2881 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2941 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3001 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3061 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3121 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3181 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3241 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     3301 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3361 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3421 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     3481 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3541 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3601 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3661 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3721 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3781 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3841 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3901 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     3961 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     4021 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     4081 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     4141 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     4201 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     4261 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     4321 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4381 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4441 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4501 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4561 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4621 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4681 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4741 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4801 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4861 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4921 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4981 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5041 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5101 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     5161 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     5221 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     5281 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     5341 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     5401 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     5461 ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc
     5521 ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag
     5581 caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa
     5641 cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta
     5701 tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg
     5761 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     5821 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     5881 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag
     5941 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag
     6001 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga
     6061 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa
     6121 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca
     6181 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac
     6241 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat
     6301 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg
     6361 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg
     6421 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
     6481 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
     6541 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
     6601 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
     6661 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
     6721 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
     6781 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
     6841 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
     6901 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
     6961 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
     7021 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
     7081 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
     7141 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     7201 aggggaccca gagagggcct atttcccatg attccttcat atttgcatat acgatacaag
     7261 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
     7321 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     7381 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     7441 ttgtggaaag gacgaggtac cggctgtgac tccaccagtt attcctcgag gaataactgg
     7501 tggagtcaca gtttttga
//
