LOCUS       sequence-213396-        7102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TRCN0000378630 (Target GATAAGTACCGTTGAGTTTG), silence human HRASLS 
            gene and express monomeric Kusabira-Orange2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7102)
  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 7102)
  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_shHRASLS.1
FEATURES             Location/Qualifiers
     source          1..7102
                     /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    2178..2295
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2344..2854
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2773..2791
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2866..3519
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             2866..3519
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     LTR             3621..3854
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3926..4047
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3963..3982)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4017..4036
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4087..4222
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4242..4261)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4243..4261)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4271..4288)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4271..4287)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4280..4302)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4429..4884
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4516..4535)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4726..4747
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4910..5014
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5015..5083,5084..5875)
                     /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(5233..5252)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6046..6634
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6535..6554
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6788..6805
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6922..6943
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6958..6963,6964..6981,6982..6988)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6996..7012
                     /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     7001..7023
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7020..7036
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7020..7036
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7055..7075
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7057..7075
                     /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 cggcgataag taccgttgag
     2101 tttgctcgag caaactcaac ggtacttatc gtttttgaat tctcgacctc gagacaaatg
     2161 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2221 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2281 aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg gccgcggctc
     2341 gagggggttg gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg
     2401 ctctgggcgt ggttccggga aacgcagcgg cgccgaccct gggtctcgca cattcttcac
     2461 gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct
     2521 tcctgctccg cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca
     2581 aacggaagcc gcacgtctca ctagtaccct cgcagacgga cagcgccagg gagcaatggc
     2641 agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag
     2701 cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt
     2761 cctgcccgcg cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc
     2821 cctcgttgac cgaatcaccg acctctctcc ccagggggat ctaccatggt gagcgtgatc
     2881 aagcccgaga tgaagatgag gtactacatg gacggctccg tcaatgggca tgagttcaca
     2941 atcgagggtg agggcacagg cagaccttac gagggacatc aggagatgac actgcgcgtc
     3001 acaatggccg agggcgggcc aatgcctttc gccttcgacc tggtgtccca cgtgttctgt
     3061 tacggccaca gagtttttac caagtaccca gaagagatcc cagactattt caagcaggcc
     3121 tttcctgagg gcctgtcctg ggagaggtcc ctggagttcg aggacggcgg ctccgcctcc
     3181 gtgagcgccc acatcagcct gaggggcaac accttctacc acaagtccaa gttcaccggc
     3241 gtgaacttcc ccgccgacgg ccccatcatg cagaaccaga gcgtggactg ggagccctcc
     3301 accgagaaga tcaccgccag cgacggcgtg ctgaagggcg acgtgaccat gtacctgaag
     3361 ctggagggcg gcggcaacca caagtgccag atgaagacca cctacaaggc cgccaaggag
     3421 atcctggaga tgcccggcga ccactacatc ggccacaggc tggtgaggaa gaccgagggc
     3481 aacatcaccg agcaggtgga ggacgccgtg gcccactccg gtggcggagg ctcgggcgga
     3541 ggtgggtcgg gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     3601 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct
     3661 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     3721 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     3781 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     3841 gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa
     3901 gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata
     3961 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4021 tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact
     4081 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4141 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     4201 tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt
     4261 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     4321 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     4381 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
     4441 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4501 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4561 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4621 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     4681 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4741 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     4801 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4861 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4921 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4981 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5041 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5101 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5161 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5221 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5281 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5341 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5401 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5461 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5521 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5581 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5641 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5701 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     5761 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5821 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     5881 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5941 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6001 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6061 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6121 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6181 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6241 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6301 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6361 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6421 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6481 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6541 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6601 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6661 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     6721 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     6781 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     6841 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     6901 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     6961 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     7021 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
     7081 aaagctggag ctgcaagctt aa
//
