LOCUS       sequence-218850-        7098 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shGLS (Target CAACTGGCCAAATTCAGTC), silence human GLS gene and 
            express monomeric Kusabira-Orange2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7098)
  AUTHORS   Quintero M, Adamoski D, Reis LMD, Ascencao CFR, Oliveira KRS, 
            Goncalves KA, Dias MM, Carazzolle MF, Dias SMG
  TITLE     Guanylate-binding protein-1 is a potential new therapeutic target 
            for triple-negative breast cancer.
  JOURNAL   BMC Cancer. 2017 Nov 7;17(1):727. doi: 10.1186/s12885-017-3726-2.
  PUBMED    29115931
REFERENCE   2  (bases 1 to 7098)
  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: "BMC 
            Cancer."; date: "2017-11-7"; volume: "17(1):727. doi"; pages: " 
            10.1186/s12885-017-3726-2"
COMMENT     Sequence Label: pLKO.1-TRC.mKO2_shGLS
FEATURES             Location/Qualifiers
     source          1..7098
                     /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..3515
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             2862..3515
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     LTR             3617..3850
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3922..4043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3959..3978)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4013..4032
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4083..4218
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4238..4257)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4239..4257)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4267..4284)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4267..4283)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4276..4298)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4425..4880
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4512..4531)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4722..4743
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4906..5010
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5011..5079,5080..5871)
                     /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(5229..5248)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6042..6630
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6531..6550
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6784..6801
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6918..6939
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6954..6959,6960..6977,6978..6984)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6992..7008
                     /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     6997..7019
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7016..7032
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7016..7032
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7051..7071
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7053..7071
                     /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 ggggatctac catggtgagc gtgatcaagc
     2881 ccgagatgaa gatgaggtac tacatggacg gctccgtcaa tgggcatgag ttcacaatcg
     2941 agggtgaggg cacaggcaga ccttacgagg gacatcagga gatgacactg cgcgtcacaa
     3001 tggccgaggg cgggccaatg cctttcgcct tcgacctggt gtcccacgtg ttctgttacg
     3061 gccacagagt ttttaccaag tacccagaag agatcccaga ctatttcaag caggcctttc
     3121 ctgagggcct gtcctgggag aggtccctgg agttcgagga cggcggctcc gcctccgtga
     3181 gcgcccacat cagcctgagg ggcaacacct tctaccacaa gtccaagttc accggcgtga
     3241 acttccccgc cgacggcccc atcatgcaga accagagcgt ggactgggag ccctccaccg
     3301 agaagatcac cgccagcgac ggcgtgctga agggcgacgt gaccatgtac ctgaagctgg
     3361 agggcggcgg caaccacaag tgccagatga agaccaccta caaggccgcc aaggagatcc
     3421 tggagatgcc cggcgaccac tacatcggcc acaggctggt gaggaagacc gagggcaaca
     3481 tcaccgagca ggtggaggac gccgtggccc actccggtgg cggaggctcg ggcggaggtg
     3541 ggtcgggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     3601 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt
     3661 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     3721 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     3781 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     3841 atctctagca gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     3901 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
     3961 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4021 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
     4081 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     4141 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     4201 gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc
     4261 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     4321 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     4381 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     4441 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4501 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4561 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4621 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4681 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4741 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4801 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4861 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4921 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4981 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5041 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5101 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5161 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5221 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5281 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5341 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5401 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5461 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5521 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5581 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5641 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5701 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5761 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5821 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5881 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5941 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6001 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6061 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6121 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6181 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6241 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6301 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6361 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6421 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6481 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6541 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6601 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6661 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     6721 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     6781 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     6841 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     6901 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     6961 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     7021 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
     7081 ctggagctgc aagcttaa
//
