LOCUS       sequence-205494-        7084 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 7084)
  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 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: "Nature. 
            2017 Nov 22. doi: 10.1038/nature24637."
COMMENT     Sequence Label: pLKO.1P shNFS1_4
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        15..263
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     15..35
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     186..205
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    360..477
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        526..1036
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     955..973
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1058..1657
                     /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(1058..1077)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1554..1574
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1785..2018
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2090..2211
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2127..2146)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2181..2200
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2251..2386
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2406..2425)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2407..2425)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2435..2452)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2435..2451)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2444..2466)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2593..3048
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2680..2699)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2890..2911
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3074..3178
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3179..3247,3248..4039)
                     /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(3397..3416)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4210..4798
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4699..4718
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4952..4969
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5086..5107
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5122..5127,5128..5145,5146..5152)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5160..5176
                     /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     5165..5187
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5184..5200
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5184..5200
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5219..5239
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5221..5239
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5267..5493
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5494..5674
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5721..5846
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6339..6572
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6757..6801
                     /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"
ORIGIN
        1 ccgccccctt caccgagggc ctatttccca tgattccttc atatttgcat atacgataca
       61 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
      121 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
      181 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
      241 tcttgtggaa aggacgaaac accgggcgca ctcttctatc aggtttctcg agaaacctga
      301 tagaagagtg cgctttttga attctcgacc tcgagacaaa tggcagtatt catccacaat
      361 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata
      421 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg
      481 gtttattaca gggacagcag agatccactt tggccgcggc tcgagggggt tggggttgcg
      541 ccttttccaa ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg
      601 gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc acgtccgttc gcagcgtcac
      661 ccggatcttc gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag
      721 tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct
      781 cactagtacc ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg
      841 ggctgtggcc aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc
      901 ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc
      961 cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac
     1021 cgacctctct ccccaggggg atccaccgga gcttaccatg accgagtaca agcccacggt
     1081 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     1141 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     1201 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     1261 cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     1321 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     1381 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     1441 cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     1501 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     1561 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     1621 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg
     1681 cccgaccgaa aggagcgcac gaccccatgc atcggtacct ttaagaccaa tgacttacaa
     1741 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     1801 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
     1861 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     1921 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     1981 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat
     2041 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat
     2101 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2161 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     2221 gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     2281 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     2341 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca
     2401 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
     2461 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     2521 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     2581 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2641 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2701 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2761 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2821 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     2881 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     2941 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3001 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     3061 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3121 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3181 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3241 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     3301 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     3361 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     3421 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     3481 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     3541 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3601 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3661 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3721 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3781 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3841 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     3901 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3961 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4021 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4081 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4141 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4201 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4261 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4321 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     4381 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4441 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4501 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4561 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4621 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4681 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4741 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4801 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4861 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4921 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4981 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     5041 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     5101 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     5161 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc
     5221 aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca
     5281 atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag
     5341 aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga
     5401 aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat
     5461 attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga
     5521 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     5581 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     5641 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
     5701 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
     5761 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
     5821 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
     5881 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
     5941 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
     6001 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     6061 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     6121 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     6181 aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa
     6241 ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag
     6301 agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc
     6361 ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga
     6421 caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa
     6481 cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct
     6541 gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc
     6601 atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt
     6661 tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata
     6721 cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa
     6781 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata
     6841 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt
     6901 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca
     6961 accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga
     7021 gacagatcca ttcgattagt gaacggatct cgacggtatc gatcacgaga ctagcctcga
     7081 gcgg
//
