LOCUS       sequence-372020-        5869 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses hMGMT together with the puromycin resistance gene via an 
            IRES..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5869)
  AUTHORS   Li J, Koczor CA, Saville KM, Hayat F, Beiser A, McClellan S, Migaud 
            ME, Sobol RW
  TITLE     Overcoming Temozolomide Resistance in Glioblastoma via Enhanced 
            NAD(+) Bioavailability and Inhibition of Poly-ADP-Ribose 
            Glycohydrolase.
  JOURNAL   Cancers (Basel). 2022 Jul 22;14(15). pii: cancers14153572. doi: 
            10.3390/cancers14153572.
  PUBMED    35892832
REFERENCE   2  (bases 1 to 5869)
  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: "Cancers 
            (Basel)."; date: "2022-07-22"; volume: "14(15). pii: 
            cancers14153572. doi"; pages: " 10.3390/cancers14153572"
COMMENT     Sequence Label: pIRES-Puro-MGMT
FEATURES             Location/Qualifiers
     source          1..5869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(83..107)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(144..163)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(145..163)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(208..411)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(237..257)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        412..791
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     963..982
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1057..1161
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1162..1230,1231..2022)
                     /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(1380..1399)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2193..2781
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2682..2701
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2935..2952
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3069..3090
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3105..3110,3111..3128,3129..3135)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3143..3159
                     /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     3148..3170
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3167..3183
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3167..3183
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3243..3262)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3309..3533
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3522..3539
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3675..4274)
                     /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(3758..3778)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4255..4274
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     misc_feature    4307..4880
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4467..4486)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     4696..4713
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(4936..4955)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          4938..5167
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     protein_bind    5267..5291
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 agctccagct tccccaaagg gctgtccagt gtggtgcgtt tcatttcaca atccttgtcc
       61 atggtgaagg gggcggccgc ggagcctgct tttttgtaca aacttgttga tatcgatccg
      121 agctcggtac caagcttggg tctccctata gtgagtcgta ttaatttcga taagccagta
      181 agcagtgggt tctctagtta gccagagagc tctgcttata tagacctccc accgtacacg
      241 cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa agtcccgttg
      301 attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg
      361 tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca ccatggtaat
      421 agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg
      481 ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat
      541 atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg
      601 tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg
      661 gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa
      721 ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat aactagtcaa
      781 taatcaatgt caacgcgtat atctggcccg tacatcgcga agcagcgcaa aacgcctaac
      841 cctaagcaga ttcttcatgc aattgtcggt caagccttgc cttgttgtag cttaaatttt
      901 gctcgcgcac tactcagcga cctccaacac acaagcaggg agcagatact ggcttaacta
      961 tgcggcatca gagcagattg tactgagagt gcaccatagg ggatcgggag atctcccgat
     1021 ccgtcgacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     1081 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     1141 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     1201 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     1261 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     1321 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     1381 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     1441 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     1501 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     1561 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     1621 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     1681 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     1741 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     1801 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     1861 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     1921 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     1981 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     2041 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     2101 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2161 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2221 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2281 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     2341 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2401 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2461 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2521 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2581 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2641 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     2701 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     2761 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     2821 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     2881 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     2941 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     3001 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     3061 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     3121 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     3181 gaccatgatt acgccaagct ctagctagag gtcgacggta tacagacatg ataagataca
     3241 ttgatgagtt tggacaaacc acaactagaa tgcactcgag ccccagctgg ttctttccgc
     3301 ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc
     3361 ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg
     3421 cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa
     3481 ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc
     3541 tgatcagcga gctctagagt cggtgggcct cgggggcggg tgcggggtcg gcggggccgc
     3601 cccgggtggc ttcggtcgga gccatggggt cgtgcgctcc tttcggtcgg gcgctgcggg
     3661 tcgtggggcg ggcgtcaggc accgggcttg cgggtcatgc accaggtgcg cggtccttcg
     3721 ggcacctcga cgtcggcggt gacggtgaag ccgagccgct cgtagaaggg gaggttgcgg
     3781 ggcgcggagg tctccaggaa ggcgggcacc ccggcgcgct cggccgcctc cactccgggg
     3841 agcacgacgg cgctgcccag acccttgccc tggtggtcgg gcgagacgcc gacggtggcc
     3901 aggaaccacg cgggctcctt gggccggtgc ggcgccagga ggccttccat ctgttgctgc
     3961 gcggccagcc gggaaccgct caactcggcc atgcgcgggc cgatctcggc gaacaccgcc
     4021 cccgcttcga cgctctccgg cgtggtccag accgccaccg cggcgccgtc gtccgcgacc
     4081 cacaccttgc cgatgtcgag cccgacgcgc gtgaggaaga gttcttgcag ctcggtgacc
     4141 cgctcgatgt ggcggtccgg gtcgacggtg tggcgcgtgg cggggtagtc ggcgaacgcg
     4201 gcggcgaggg tgcgtacggc ccgggggacg tcgtcgcggg tggcgaggcg caccgtgggc
     4261 ttgtactcgg tcatggaagg tcgtctcctt gtgggttgtg gcaagcttat catcgtgttt
     4321 ttcaaaggaa aaccacgtcc ccgtggttcg gggggcctag acgttttttt aacctcgact
     4381 aaacacatgt aaagcatgtg caccgaggcc ccagatcaga tcccatacaa tggggtacct
     4441 tctgggcatc cttcagcccc ttgttgaata cgcttgagga gagccatttg actctttcca
     4501 caactatcca actcacaacg tggcactggg gttgtgccgc ctttgcaggt gtatcttata
     4561 cacgtggctt ttggccgcag aggcacctgt cgccaggtgg ggggttccgc tgcctgcaaa
     4621 gggtcgctac agacgttgtt tgtcttcaag aagcttccag aggaactgct tccttcacga
     4681 cattcaacag accttgcatt cctttggcga gaggggaaag acccctagga atgctcgtca
     4741 agaagacagg gccaggtttc cgggccctca cattgccaaa agacggcaat atggtggaaa
     4801 ataacatata gacaaacgca caccggcctt attccaagcg gcttcggcca gtaacgttag
     4861 gggsgggggg gggagagggg cggaattggc cgccctagat gcatgctcga cctgcagttg
     4921 gacctgggag tggacacctg tggagagaaa ggcaaagtgg atgtcattgt cactcaagtg
     4981 tatggccaga tctcaagcct gccacacctc aagcttgaca acaaaaagat tgtcttttct
     5041 gaccagatgg acgcggccac cctcaaaggc atcaccgcgg gccaggtgaa tatcaaatcc
     5101 tcctcgtttt tggaaactga caatcttagc gcagaagtca tgcccgcttt tgagagggag
     5161 tactcacccc aacagctggc cctcgcagac agcgaattaa ttccagcaca ctggcggccg
     5221 ttactagtgg atccactgaa ttccgtcgac gcggccgcag atatcaacca ctttgtacaa
     5281 gaaagctggg tcggcgcgcc cacccttcta gtttcggcca gcaggcgggg agcccgaggt
     5341 agctcccgct cccttgagcc aggcccctgc cagacctgag ctccctccca agcctggctt
     5401 ccccaaccgg tggccttcat gggccagaag ccattccttc acggccagtc ctccggagta
     5461 gttgcccacg gctccgctgc tgcagaccac tctgtggcac gggatgagga tggggacagg
     5521 attgcctctc attgctcctc ccactgctcg cgcggctttg gggttgcctg ccagggctgc
     5581 taattgctgg taagaaatca cttctccgaa tttcacaacc ttcagcagct tccataacac
     5641 ctgtctggtg aacgactctt gctggaaaac gggatggtga agagccggca cggggaactc
     5701 ttcgatagcc tcgggctggt ggaaataggc attcagccag gctgtgcact gcatcagggg
     5761 ctccggacct ccgagaaccg cagcgggggc tgggacctcc acggcatcag ctgcagacgt
     5821 ccccttgccc aggagcttta tttcgtgcag accctgctca caaccagac
//
