LOCUS       sequence-198379-        7518 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Suppression of ISCU.
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 shISCU
FEATURES             Location/Qualifiers
     source          1..7518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    41..158
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        207..717
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     636..654
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             739..1338
                     /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(739..758)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1235..1255
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1354..1942
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1407..1427)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1825..1836)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2014..2247
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2325..2446
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2362..2381)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2416..2435
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2486..2621
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2641..2660)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2642..2660)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2670..2687)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2670..2686)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2679..2701)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2828..3283
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2915..2934)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3125..3146
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3309..3413
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3414..3482,3483..4274)
                     /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(3632..3651)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4445..5033
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4934..4953
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5187..5204
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5321..5342
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5357..5362,5363..5380,5381..5387)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5395..5411
                     /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     5400..5422
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5419..5435
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5419..5435
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5454..5474
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5456..5474
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5502..5728
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5729..5909
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5956..6081
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6574..6807
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6992..7036
                     /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        7214..7462
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7214..7234
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7385..7404
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
       61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
      121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
      181 gagatccact ttggcgccgg ctcgaggggg cccgggttgc gccttttcca aggcagccct
      241 gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac
      301 cctgggtctc gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc
      361 cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg
      421 ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac
      481 ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc
      541 tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt
      601 ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg
      661 gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagggg
      721 gatccaccgg agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
      781 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
      841 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
      901 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgccgtggcg
      961 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     1021 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     1081 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     1141 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     1201 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     1261 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     1321 cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac ctctggatta caaaatttgt
     1381 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     1441 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     1501 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     1561 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     1621 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     1681 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     1741 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     1801 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     1861 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     1921 tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag
     1981 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
     2041 gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg
     2101 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     2161 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     2221 tttagtcagt gtggaaaatc tctagcagta cgtatagtag ttcatgtcat cttattattc
     2281 agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag
     2341 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2401 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct
     2461 agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     2521 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     2581 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg
     2641 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     2701 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     2761 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     2821 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2881 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2941 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     3001 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     3061 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3121 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3181 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3241 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     3301 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3361 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     3421 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     3481 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     3541 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     3601 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     3661 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     3721 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     3781 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3841 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3901 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     3961 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4021 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4081 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4141 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4201 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4261 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4321 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4381 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4441 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4501 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4561 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     4621 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4681 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4741 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4801 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4861 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4921 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4981 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5041 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5101 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     5161 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     5221 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     5281 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     5341 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     5401 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
     5461 accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact
     5521 cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa
     5581 gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca
     5641 acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt
     5701 atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg
     5761 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
     5821 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
     5881 cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa
     5941 gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga
     6001 ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag
     6061 agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa
     6121 attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc
     6181 agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga
     6241 caaatactgg gacagctaca accatccctt cagacaggat cagaagaact tagatcatta
     6301 tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag
     6361 gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg
     6421 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     6481 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     6541 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     6601 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     6661 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     6721 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     6781 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     6841 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     6901 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     6961 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     7021 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     7081 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     7141 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     7201 gaggggaccc agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     7261 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     7321 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     7381 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     7441 cttgtggaaa ggacgaggta ccgggtccct tgacaagaca tctaactcga gttagatgtc
     7501 ttgtcaaggg actttttg
//
