LOCUS       sequence-169780-        7102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TRCN0000116119 (Target CGACGAAAGGCATGTACCATA) for silencing GBP1 
            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-shGBP1.1.mKO2
FEATURES             Location/Qualifiers
     source          1..7102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..27
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     241..263
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     255..272
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     256..272
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     282..301
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        282..300
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      321..456
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    496..617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(507..526)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     561..580
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             689..922
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             complement(1024..1677)
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             complement(1024..1677)
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        complement(1689..2199)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1752..1770)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    2248..2365
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(2462..2710)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2520..2539)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2690..2710)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(3008..3052)
                     /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"
     misc_feature    3237..3470
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    3963..4088
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             4135..4315
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(4316..4542)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(4570..4590)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4570..4588)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4609..4625)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4609..4625)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4622..4644)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4633..4649
                     /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)."
     promoter        complement(join(4657..4663,4664..4681,4682..4687))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4702..4723
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4840..4857)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5011..5599)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5091..5110)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5770..6561,6562..6630))
                     /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     6393..6412
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6631..6735)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(6761..7102,1..114))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6898..6919)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
       61 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
      121 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
      181 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
      241 cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg
      301 gcgaattggg tacgtcccta ggcctccaaa aaagcctcct cactacttct ggaatagctc
      361 agaggccgag gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg
      421 agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggatagc
      481 tagagccaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
      541 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
      601 gctgcaataa acaagttcct ctcactctct gatattcatt tctttgcaag ttataaatac
      661 tgaataataa gatgacatga actactactg ctagagattt tccacactga ctaaaagggt
      721 ctgagggatc tctagttacc agagtcacac aacagacggg cacacactac ttgaagcact
      781 caaggcaagc tttattgagg cttaagcagt gggttcccta gttagccaga gagctcccag
      841 gctcagatct ggtctaacca gagagaccca gtacaagcaa aaagcagatc ttgtcttcgt
      901 tgggagtgaa ttagcccttc cagtcccccc ttttctttta aaaagtggct aagatctaca
      961 gctgccttgt aagtcattgg tcttaaaggt acccgaccca cctccgcccg agcctccgcc
     1021 accggagtgg gccacggcgt cctccacctg ctcggtgatg ttgccctcgg tcttcctcac
     1081 cagcctgtgg ccgatgtagt ggtcgccggg catctccagg atctccttgg cggccttgta
     1141 ggtggtcttc atctggcact tgtggttgcc gccgccctcc agcttcaggt acatggtcac
     1201 gtcgcccttc agcacgccgt cgctggcggt gatcttctcg gtggagggct cccagtccac
     1261 gctctggttc tgcatgatgg ggccgtcggc ggggaagttc acgccggtga acttggactt
     1321 gtggtagaag gtgttgcccc tcaggctgat gtgggcgctc acggaggcgg agccgccgtc
     1381 ctcgaactcc agggacctct cccaggacag gccctcagga aaggcctgct tgaaatagtc
     1441 tgggatctct tctgggtact tggtaaaaac tctgtggccg taacagaaca cgtgggacac
     1501 caggtcgaag gcgaaaggca ttggcccgcc ctcggccatt gtgacgcgca gtgtcatctc
     1561 ctgatgtccc tcgtaaggtc tgcctgtgcc ctcaccctcg attgtgaact catgcccatt
     1621 gacggagccg tccatgtagt acctcatctt catctcgggc ttgatcacgc tcaccatggt
     1681 agatccccct ggggagagag gtcggtgatt cggtcaacga gggagccgac tgccgacgtg
     1741 cgctccggag gcttgcagaa tgcggaacac cgcgcgggca ggaacagggc ccacactacc
     1801 gccccacacc ccgcctcccg caccgcccct tcccggccgc tgctctcggc gcgccctgct
     1861 gagcagccgc tattggccac agcccatcgc ggtcggcgcg ctgccattgc tccctggcgc
     1921 tgtccgtctg cgagggtact agtgagacgt gcggcttccg tttgtcacgt ccggcacgcc
     1981 gcgaaccgca aggaaccttc ccgacttagg ggcggagcag gaagcgtcgc cggggggccc
     2041 acaagggtag cggcgaagat ccgggtgacg ctgcgaacgg acgtgaagaa tgtgcgagac
     2101 ccagggtcgg cgccgctgcg tttcccggaa ccacgcccag agcagccgcg tccctgcgca
     2161 aacccagggc tgccttggaa aaggcgcaac cccaaccccc tcgagccgcg gccaaagtgg
     2221 atctctgctg tccctgtaat aaacccgaaa attttgaatt tttgtaattt gtttttgtaa
     2281 ttctttagtt tgtatgtctg ttgctattat gtctactatt ctttcccctg cactgtaccc
     2341 cccaatcccc ccttttcttt taaaattgtg gatgaatact gccatttgtc tcgaggtcga
     2401 gaattcaaaa acgacgaaag gcatgtacca tactcgagta tggtacatgc ctttcgtcgc
     2461 cggtgtttcg tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta
     2521 cggtaagcat atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa
     2581 attattactt tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa
     2641 ttccaattat ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa
     2701 ataggccctc ggtgaagggg gcggccgctc gaggctagtc tcgtgatcga taccgtcgag
     2761 atccgttcac taatcgaatg gatctgtctc tgtctctctc tccaccttct tcttctattc
     2821 cttcgggcct gtcgggtccc ctcggggttg ggaggtgggt ctgaaacgat aatggtgaat
     2881 atccctgcct aactctattc actatagaaa gtacagcaaa aactattctt aaacctacca
     2941 agcctcctac tatcattatg aataatttta tataccacag ccaatttgtt atgttaaacc
     3001 aattccacaa acttgcccat ttatctaatt ccaataattc ttgttcattc ttttcttgct
     3061 ggttttgcga ttcttcaatt aaggagtgta ttaagcttgt gtaattgtta atttctctgt
     3121 cccactccat ccaggtcgtg tgattccaaa tctgttccag agatttatta ctccaactag
     3181 cattccaagg cacagcagtg gtgcaaatga gttttccaga gcaaccccaa atccccagga
     3241 gctgttgatc ctttaggtat ctttccacag ccaggattct tgcctggagc tgcttgatgc
     3301 cccagactgt gagttgcaac agatgctgtt gcgcctcaat agccctcagc aaattgttct
     3361 gctgctgcac tataccagac aataattgtc tggcctgtac cgtcagcgtc attgacgctg
     3421 cgcccatagt gcttcctgct gctcccaaga acccaaggaa caaagctcct attcccactg
     3481 ctcttttttc tctctgcacc actcttctct ttgccttggt gggtgctact cctaatggtt
     3541 caatttttac tactttatat ttatataatt cacttctcca attgtccctc atatctcctc
     3601 ctccaggtct gaagatcagc ggccgcttgc tgtgcggtgg tcttactttt gttttgctct
     3661 tcctctatct tgtctaaagc ttccttggtg tcttttatct ctatcctttg atgcacacaa
     3721 tagagggttg ctactgtatt atataatgat ctaagttctt ctgatcctgt ctgaagggat
     3781 ggttgtagct gtcccagtat ttgtctacag ccttctgatg tttctaacag gccaggatta
     3841 actgcgaatc gttctagctc cctgcttgcc catactatat gttttaattt atattttttc
     3901 tttccccctg gccttaaccg aattttttcc catcgcgatc taattctccc ccgcttaata
     3961 ctgacgctct cgcacccatc tctctccttc tagcctccgc tagtcaaaat ttttggcgta
     4021 ctcaccagtc gccgcccctc gcctcttgcc gtgcgcgctt cagcaagccg agtcctgcgt
     4081 cgagagagct cctctggttt ccctttcgct ttcaagtccc tgttcgggcg ccactgctag
     4141 agattttcca cactgactaa aagggtctga gggatctcta gttaccagag tcacacaaca
     4201 gacgggcaca cactacttga agcactcaag gcaagcttta ttgaggctta agcagtgggt
     4261 tccctagtta gccagagagc tcccaggctc agatctggtc taaccagaga gacccgttta
     4321 tgtatcgagc taggcactta aatacaatat ctctgcaatg cggcaattca gtggttcgtc
     4381 caatccatgt cagacccgtc tgttgccttc ctaataaggc acgatcgtac caccttactt
     4441 ccaccaatcg gcatgcacgg tgctttttct ctccttgtaa ggcatgttgc taactcatcg
     4501 ttaccatgtt gcaagactac aagagtattg cataagacta cattaagctt gcagctccag
     4561 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     4621 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4681 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4741 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4801 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4861 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4921 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4981 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5041 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5101 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5161 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5221 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5281 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5341 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5401 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5461 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5521 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5581 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5641 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5701 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5761 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5821 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5881 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5941 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6001 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6061 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6121 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6181 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6241 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6301 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6361 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6421 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6481 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6541 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6601 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6661 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6721 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     6781 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     6841 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     6901 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     6961 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     7021 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     7081 ggaaagccgg cgaacgtggc ga
//
