LOCUS       sequence-207665-        7102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TRCN0000002620 (Target GCGATAAGTACCGTTGAGTTT), 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.2
FEATURES             Location/Qualifiers
     source          1..7102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     426..444
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             519..1172
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             519..1172
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     LTR             1274..1507
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1579..1700
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1616..1635)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1670..1689
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1740..1875
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1895..1914)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1896..1914)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1924..1941)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1924..1940)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1933..1955)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2082..2537
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2169..2188)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2379..2400
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2563..2667
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2668..2736,2737..3528)
                     /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(2886..2905)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3699..4287
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4188..4207
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4441..4458
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4575..4596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4611..4616,4617..4634,4635..4641)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4649..4665
                     /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     4654..4676
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4673..4689
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4673..4689
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4708..4728
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4710..4728
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4756..4982
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             4983..5163
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5210..5335
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5828..6061
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6246..6290
                     /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        6588..6836
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6588..6608
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6759..6778
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    6933..7050
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(7099..7102,1..507)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
ORIGIN
        1 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
       61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt
      121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
      181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
      241 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
      301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
      361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
      421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
      481 gaccgaatca ccgacctctc tccccagggg gatctaccat ggtgagcgtg atcaagcccg
      541 agatgaagat gaggtactac atggacggct ccgtcaatgg gcatgagttc acaatcgagg
      601 gtgagggcac aggcagacct tacgagggac atcaggagat gacactgcgc gtcacaatgg
      661 ccgagggcgg gccaatgcct ttcgccttcg acctggtgtc ccacgtgttc tgttacggcc
      721 acagagtttt taccaagtac ccagaagaga tcccagacta tttcaagcag gcctttcctg
      781 agggcctgtc ctgggagagg tccctggagt tcgaggacgg cggctccgcc tccgtgagcg
      841 cccacatcag cctgaggggc aacaccttct accacaagtc caagttcacc ggcgtgaact
      901 tccccgccga cggccccatc atgcagaacc agagcgtgga ctgggagccc tccaccgaga
      961 agatcaccgc cagcgacggc gtgctgaagg gcgacgtgac catgtacctg aagctggagg
     1021 gcggcggcaa ccacaagtgc cagatgaaga ccacctacaa ggccgccaag gagatcctgg
     1081 agatgcccgg cgaccactac atcggccaca ggctggtgag gaagaccgag ggcaacatca
     1141 ccgagcaggt ggaggacgcc gtggcccact ccggtggcgg aggctcgggc ggaggtgggt
     1201 cgggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     1261 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct
     1321 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     1381 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     1441 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     1501 tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga
     1561 atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     1621 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     1681 aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca
     1741 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     1801 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     1861 gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg
     1921 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     1981 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     2041 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     2101 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2161 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2221 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2281 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     2341 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2401 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2461 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     2521 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2581 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     2641 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     2701 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     2761 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     2821 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2881 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     2941 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     3001 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     3061 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     3121 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     3181 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     3241 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     3301 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     3361 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     3421 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     3481 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     3541 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     3601 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3661 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3721 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3781 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3841 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3901 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3961 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4021 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4081 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4141 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4201 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4261 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4321 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4381 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4441 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     4501 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     4561 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     4621 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     4681 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
     4741 gagctgcaag cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga
     4801 tgagttagca acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag
     4861 taaggtggta cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac
     4921 gaaccactga attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa
     4981 acgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     5041 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     5101 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     5161 gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg
     5221 caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac
     5281 gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat
     5341 taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa
     5401 aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa
     5461 tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc
     5521 ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg
     5581 tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga
     5641 gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag
     5701 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac
     5761 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag
     5821 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag
     5881 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga
     5941 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca
     6001 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc
     6061 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta
     6121 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca
     6181 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc
     6241 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt
     6301 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg
     6361 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt
     6421 caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa
     6481 tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg aacggatctc
     6541 gacggtatcg atcacgagac tagcctcgag cggccgcccc cttcaccgag ggcctatttc
     6601 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     6661 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     6721 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     6781 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgggc
     6841 gataagtacc gttgagtttc tcgagaaact caacggtact tatcgctttt tgaattctcg
     6901 acctcgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     6961 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     7021 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     7081 ctttggccgc ggctcgaggg gg
//
