LOCUS       sequence-169779-        7101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shLuc (Target TTACGCTGAGTACTTCGA) for silencing luciferase gene as a
            control and express monomeric Kusabira-Orange2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7101)
  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 7101)
  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-shLuc.mKO2
FEATURES             Location/Qualifiers
     source          1..7101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..233
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             234..414
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    461..586
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1079..1312
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1497..1541
                     /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        1839..2087
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1839..1859
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2010..2029
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2183..2300
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2349..2859
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2778..2796
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2871..3524
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             2871..3524
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     LTR             3626..3859
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3931..4052
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3968..3987)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4022..4041
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4092..4227
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4247..4266)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4248..4266)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4276..4293)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4276..4292)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4285..4307)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4434..4889
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4521..4540)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4731..4752
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4915..5019
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5020..5088,5089..5880)
                     /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(5238..5257)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6051..6639
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6540..6559
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6793..6810
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6927..6948
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6963..6968,6969..6986,6987..6993)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7001..7017
                     /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     7006..7028
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7025..7041
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7025..7041
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7060..7080
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7062..7080
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc
       61 aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt
      121 acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg
      181 aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc
      241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      361 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
      421 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
      481 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
      541 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
      601 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
      661 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
      721 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
      781 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
      841 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
      901 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga
      961 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag
     1021 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag
     1081 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga
     1141 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc
     1201 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc
     1261 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt
     1321 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta
     1381 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta
     1441 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga
     1501 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa
     1561 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
     1621 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
     1681 cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag
     1741 aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatct cgacggtatc
     1801 gatcacgaga ctagcctcga gcggccgccc ccttcaccga gggcctattt cccatgattc
     1861 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     1921 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     1981 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     2041 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggc ttacgctgag
     2101 tacttcgacc tgacccatcg aagtactcag cgtaagtttt tgaattctcg acctcgagac
     2161 aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag
     2221 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa
     2281 ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggccgc
     2341 ggctcgaggg ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc
     2401 ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc
     2461 ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg
     2521 acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg
     2581 tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca
     2641 atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc
     2701 gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc
     2761 ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc
     2821 ggctccctcg ttgaccgaat caccgacctc tctccccagg gggatctacc atggtgagcg
     2881 tgatcaagcc cgagatgaag atgaggtact acatggacgg ctccgtcaat gggcatgagt
     2941 tcacaatcga gggtgagggc acaggcagac cttacgaggg acatcaggag atgacactgc
     3001 gcgtcacaat ggccgagggc gggccaatgc ctttcgcctt cgacctggtg tcccacgtgt
     3061 tctgttacgg ccacagagtt tttaccaagt acccagaaga gatcccagac tatttcaagc
     3121 aggcctttcc tgagggcctg tcctgggaga ggtccctgga gttcgaggac ggcggctccg
     3181 cctccgtgag cgcccacatc agcctgaggg gcaacacctt ctaccacaag tccaagttca
     3241 ccggcgtgaa cttccccgcc gacggcccca tcatgcagaa ccagagcgtg gactgggagc
     3301 cctccaccga gaagatcacc gccagcgacg gcgtgctgaa gggcgacgtg accatgtacc
     3361 tgaagctgga gggcggcggc aaccacaagt gccagatgaa gaccacctac aaggccgcca
     3421 aggagatcct ggagatgccc ggcgaccact acatcggcca caggctggtg aggaagaccg
     3481 agggcaacat caccgagcag gtggaggacg ccgtggccca ctccggtggc ggaggctcgg
     3541 gcggaggtgg gtcgggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     3601 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     3661 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     3721 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     3781 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     3841 gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt
     3901 gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac
     3961 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4021 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc
     4081 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4141 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     4201 agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc
     4261 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     4321 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     4381 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     4441 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4501 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4561 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4621 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     4681 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4741 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     4801 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4861 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     4921 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4981 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5041 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5101 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5161 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5221 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5281 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5341 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5401 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5461 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5521 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5581 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5641 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5701 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5761 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5821 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5881 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     5941 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6001 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6061 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6121 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6181 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6241 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6301 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6361 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6421 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6481 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6541 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6601 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6661 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6721 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     6781 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     6841 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     6901 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     6961 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     7021 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
     7081 gaacaaaagc tggagctgca a
//
