LOCUS       sequence-213701-        7080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shGLS (Target CAACTGGCCAAATTCAGTC), silence human GLS gene, 
            puromycin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7080)
  AUTHORS   Dias MM, Adamoski D, Dos Reis LM, Ascencao CFR, de Oliveira KRS, 
            Mafra ACP, da Silva Bastos AC, Quintero M, de G Cassago C, Ferreira 
            IM, Fidelis CHV, Rocco SA, Bajgelman MC, Stine Z, Berindan-Neagoe I,
            Calin GA, Ambrosio ALB, Dias SMG
  TITLE     GLS2 is protumorigenic in breast cancers.
  JOURNAL   Oncogene. 2020 Jan;39(3):690-702. doi: 10.1038/s41388-019-1007-z. 
            Epub 2019 Sep 20.
  PUBMED    31541193
REFERENCE   2  (bases 1 to 7080)
  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"; doi: 
            "10.1038/s41388-019-1007-z"; journalName: "Oncogene"; date: 
            "2020-01"; volume: "39"; issue: "3"; pages: "690-702"
COMMENT     Sequence Label: pLKO.puro_shGLS
FEATURES             Location/Qualifiers
     source          1..7080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /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        1833..2081
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1833..1853
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2004..2023
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2174..2291
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2340..2850
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2769..2787
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2872..3471
                     /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(2872..2891)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3368..3388
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3599..3832
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3904..4025
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3941..3960)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3995..4014
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4065..4200
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4220..4239)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4221..4239)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4249..4266)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4249..4265)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4258..4280)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4407..4862
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4494..4513)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4704..4725
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4888..4992
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4993..5061,5062..5853)
                     /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(5211..5230)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6024..6612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6513..6532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6766..6783
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6900..6921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6936..6941,6942..6959,6960..6966)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6974..6990
                     /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     6979..7001
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6998..7014
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6998..7014
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7033..7053
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7035..7053
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     1741 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac
     1801 gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat
     1861 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     1921 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     1981 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2041 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggcaactgg ccaaattcag
     2101 tcctcgagga ctgaatttgg ccagttgttt ttgaattctc gacctcgaga caaatggcag
     2161 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     2221 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     2281 ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg
     2341 gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct
     2401 gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc
     2461 gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct
     2521 gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg
     2581 gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg
     2641 cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc
     2701 ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg
     2761 cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc
     2821 gttgaccgaa tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag
     2881 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     2941 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     3001 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     3061 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     3121 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     3181 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     3241 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     3301 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     3361 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     3421 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc
     3481 cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga
     3541 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     3601 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     3661 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     3721 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     3781 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt
     3841 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag
     3901 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3961 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4021 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc
     4081 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4141 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4201 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     4261 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     4321 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     4381 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     4441 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     4501 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4561 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4621 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4681 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     4741 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4801 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     4861 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4921 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4981 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5041 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5101 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5161 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5221 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5281 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5341 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5401 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5461 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5521 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5581 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5641 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5701 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5761 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5821 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5881 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5941 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6001 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6061 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6121 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6181 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6241 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6301 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6361 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6421 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6481 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6541 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6601 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6661 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6721 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6781 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     6841 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     6901 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     6961 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     7021 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa
//
