LOCUS       sequence-153942-        8551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PCYT2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8551)
  AUTHORS   Chidley C, Trauger SA, Birsoy K, O'Shea EK
  TITLE     The anticancer natural product ophiobolin A induces cytotoxicity by 
            covalent modification of phosphatidylethanolamine.
  JOURNAL   Elife. 2016 Jul 12;5. pii: e14601. doi: 10.7554/eLife.14601.
  PUBMED    27403889
REFERENCE   2  (bases 1 to 8551)
  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: "Elife."; 
            date: "2016-07-12"; volume: "5. pii: e14601. doi"; pages: " 
            10.7554/eLife.14601"
COMMENT     Sequence Label: pLJM1-PCYT2
FEATURES             Location/Qualifiers
     source          1..8551
                     /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"
     LTR             1473..1706
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1778..1899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1815..1834)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1869..1888
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1939..2074
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2094..2113)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2095..2113)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2123..2140)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2123..2139)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2132..2154)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2281..2736
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2368..2387)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2578..2599
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2762..2866
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2867..2935,2936..3727)
                     /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(3085..3104)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3898..4486
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4387..4406
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4640..4657
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4774..4795
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4810..4815,4816..4833,4834..4840)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4848..4864
                     /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     4853..4875
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4872..4888
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4872..4888
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4907..4927
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4909..4927
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4955..5181
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5182..5362
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5409..5534
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6027..6260
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6445..6489
                     /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"
     primer_bind     6787..6807
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        7096..7299
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7250..7270
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             7340..7363
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
ORIGIN
        1 atcttcgaat tctcgacctc gagacaaatg gcagtattca tccacaattt taaaagaaaa
       61 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
      121 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
      181 gacagcagag atccactttg gccgcggctc gagggggttg 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 ggtggcggtc 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 cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag
     1381 gagcgcacga ccccatgcat cggtaccttt aagaccaatg acttacaagg cagctgtaga
     1441 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     1501 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     1561 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     1621 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     1681 ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta ttcagtattt
     1741 ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa
     1801 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     1861 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc
     1921 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     1981 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     2041 ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata
     2101 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     2161 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     2221 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg
     2281 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2341 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2401 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2461 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2521 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2581 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2641 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2701 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     2761 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2821 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     2881 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     2941 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3001 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3061 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     3121 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3181 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3241 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3301 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3361 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     3421 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3481 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3541 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3601 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3661 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3721 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3781 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     3841 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3901 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3961 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4021 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     4081 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4141 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4201 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4261 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4321 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4381 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4441 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4501 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4561 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4621 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     4681 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     4741 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     4801 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     4861 acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca
     4921 ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt cttatgcaat actcttgtag
     4981 tcttgcaaca tggtaacgat gagttagcaa catgccttac aaggagagaa aaagcaccgt
     5041 gcatgccgat tggtggaagt aaggtggtac gatcgtgcct tattaggaag gcaacagacg
     5101 ggtctgacat ggattggacg aaccactgaa ttgccgcatt gcagagatat tgtatttaag
     5161 tgcctagctc gatacataaa cgggtctctc tggttagacc agatctgagc ctgggagctc
     5221 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     5281 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     5341 tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac
     5401 cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg
     5461 ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg
     5521 ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt
     5581 taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc
     5641 tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac
     5701 tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata
     5761 cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt
     5821 tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg
     5881 atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat
     5941 aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg
     6001 cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca
     6061 ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct
     6121 ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg
     6181 caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac
     6241 ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact
     6301 gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg
     6361 acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt
     6421 gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg
     6481 gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa attattcata
     6541 atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat
     6601 agagttaggc agggatattc accattatcg tttcagaccc acctcccaac cccgagggga
     6661 cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt
     6721 cgattagtga acggatctcg acggtatcga tcacgagact agcctcgagc ggccgccccc
     6781 ttcaccgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     6841 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     6901 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     6961 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     7021 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     7081 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
     7141 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
     7201 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
     7261 ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc
     7321 cgctagcgct accggtatgg actacaagga tgacgatgac aagggtggtg gtggtggtgc
     7381 gatgatccgg aacgggcgcg gggctgcagg cggcgcagag cagccgggcc cggggggcag
     7441 gcgcgccgtg agggtgtggt gcgatggctg ctatgacatg gtgcattacg gccactccaa
     7501 ccagctgcgc caggcacggg ccatgggtga ctacctcatc gtaggcgtgc acaccgatga
     7561 ggagatcgcc aagcacaagg ggcccccggt gttcactcag gaggagagat acaagatggt
     7621 gcaggccatc aaatgggtgg acgaggtggt gccagcggct ccctacgtca ctacactaga
     7681 gaccctggac aaatacaact gtgacttctg tgttcacggc aatgacatca ccctgactgt
     7741 agatggccgg gacacctatg aggaagtaaa gcaggctggg aggtacagag aatgcaagcg
     7801 cacgcaaggg gtgtccacca cagacctcgt gggccgcatg ctgctggtaa ccaaagccca
     7861 tcacagcagc caggagatgt cctctgagta ccgggagtat gcagacagtt ttggcaagtg
     7921 ccctggtggg cggaacccct ggaccggggt atcccagttc ctgcagacat ctcagaagat
     7981 catccagttt gcttctggga aggagcccca gccaggggag acagtcatct atgtggctgg
     8041 tgccttcgac ctgttccaca tcgggcatgt ggacttcctg gagaaggtgc acaggctggc
     8101 agagaggccc tacatcatcg cgggcttaca ctttgaccag gaggtcaatc actacaaggg
     8161 gaagaactac cccatcatga atctgcatga acggactctg agcgtgctgg cctgccggta
     8221 cgtgtcagaa gtggtgattg gagccccgta cgcggtcaca gcagagctcc taagtcactt
     8281 caaggtggac ctggtgtgtc acggcaagac agaaattatc cctgacaggg atggctccga
     8341 cccataccag gagcccaaga gaaggggcat cttccgtcag attgacagtg gcagcaacct
     8401 caccacagac ctcatcgtcc agcggatcat caccaacagg ttggagtatg aggcgcgaaa
     8461 ccagaagaag gaagccaagg agctggcctt cctggaggct gccaggcagc aggcggcaca
     8521 gcccctgggg gagcgcgatg gtgacttcta a
//
