LOCUS       sequence-218291-        6813 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shGAC (Target CCTCTGTTCTGTCAGAGTT), silence glutaminase isoform GAC,
            blasticidin selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6813)
  AUTHORS   Sandra Martha Gomes Dias
  TITLE     HuR (ELAVL1) silencing sensitizes triple-negative breast cancer to 
            glutaminase-targeted therapy
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6813)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pLKO.1-blast shGAC
FEATURES             Location/Qualifiers
     source          1..6813
                     /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             2862..3260
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             3332..3565
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3637..3758
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3674..3693)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3728..3747
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3798..3933
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3953..3972)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3954..3972)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3982..3999)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3982..3998)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3991..4013)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4140..4595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4227..4246)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4437..4458
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4621..4725
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4726..4794,4795..5586)
                     /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(4944..4963)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5757..6345
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6246..6265
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6499..6516
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6633..6654
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6669..6674,6675..6692,6693..6699)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6707..6723
                     /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     6712..6734
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6731..6747
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6731..6747
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6766..6786
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6768..6786
                     /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 cggcctctgt tctgtcagag
     2101 ttctcgagaa ctctgacaga acagaggttt 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 catggccaag cctttgtctc
     2881 aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg
     2941 aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca
     3001 atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg
     3061 ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct
     3121 tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca
     3181 tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg
     3241 gttatgtgtg ggagggctaa ggtaccttta agaccaatga cttacaaggc agctgtagat
     3301 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     3361 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     3421 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     3481 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     3541 tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta
     3601 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat
     3661 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     3721 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc
     3781 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3841 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     3901 tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag
     3961 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     4021 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     4081 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     4141 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4201 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4261 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4321 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     4381 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4441 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4501 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4561 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     4621 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     4681 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     4741 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4801 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     4861 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     4921 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     4981 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5041 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5101 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5161 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5221 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5281 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5341 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5401 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     5461 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     5521 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5581 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     5641 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5701 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5761 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5821 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5881 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     5941 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6001 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6061 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6121 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6181 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6241 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6301 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6361 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6421 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     6481 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     6541 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     6601 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     6661 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     6721 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
     6781 taaagggaac aaaagctgga gctgcaagct taa
//
