LOCUS       sequence-169782-        7102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TRCN0000116120 (Target TGAGACGACGAAAGGCATGTA) 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-shGBP1.2.mKO2
FEATURES             Location/Qualifiers
     source          1..7102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(87..675)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(167..186)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(846..1637,1638..1706))
                     /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     1469..1488
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1707..1811)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1837..2292)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1974..1995)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2186..2205
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2419..2441
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2433..2450
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2434..2450
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2460..2479
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2460..2478
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2499..2634
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2674..2795
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2685..2704)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2739..2758
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             2867..3100
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             complement(3202..3855)
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             complement(3202..3855)
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        complement(3867..4377)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3930..3948)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    4426..4543
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(4640..4888)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4698..4717)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4868..4888)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(5186..5230)
                     /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    5415..5648
                     /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    6141..6266
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6313..6493
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(6494..6720)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(6748..6768)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6748..6766)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6787..6803)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6787..6803)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6800..6822)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6811..6827
                     /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(6835..6841,6842..6859,6860..6865))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6880..6901
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7018..7035)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
       61 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
      121 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
      181 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
      241 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
      301 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
      361 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
      421 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
      481 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
      541 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
      601 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
      661 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
      721 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
      781 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
      841 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
      901 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
      961 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     1021 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     1081 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     1141 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     1201 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     1261 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     1321 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     1381 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     1441 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     1501 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     1561 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     1621 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     1681 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     1741 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     1801 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     1861 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     1921 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     1981 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     2041 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     2101 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     2161 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     2221 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     2281 tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     2341 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     2401 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt
     2461 aatacgactc actatagggc gaattgggta cgtccctagg cctccaaaaa agcctcctca
     2521 ctacttctgg aatagctcag aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat
     2581 tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg
     2641 agttaggggc gggatagcta gagccagaca tgataagata cattgatgag tttggacaaa
     2701 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     2761 tatttgtaac cattataagc tgcaataaac aagttcctct cactctctga tattcatttc
     2821 tttgcaagtt ataaatactg aataataaga tgacatgaac tactactgct agagattttc
     2881 cacactgact aaaagggtct gagggatctc tagttaccag agtcacacaa cagacgggca
     2941 cacactactt gaagcactca aggcaagctt tattgaggct taagcagtgg gttccctagt
     3001 tagccagaga gctcccaggc tcagatctgg tctaaccaga gagacccagt acaagcaaaa
     3061 agcagatctt gtcttcgttg ggagtgaatt agcccttcca gtcccccctt ttcttttaaa
     3121 aagtggctaa gatctacagc tgccttgtaa gtcattggtc ttaaaggtac ccgacccacc
     3181 tccgcccgag cctccgccac cggagtgggc cacggcgtcc tccacctgct cggtgatgtt
     3241 gccctcggtc ttcctcacca gcctgtggcc gatgtagtgg tcgccgggca tctccaggat
     3301 ctccttggcg gccttgtagg tggtcttcat ctggcacttg tggttgccgc cgccctccag
     3361 cttcaggtac atggtcacgt cgcccttcag cacgccgtcg ctggcggtga tcttctcggt
     3421 ggagggctcc cagtccacgc tctggttctg catgatgggg ccgtcggcgg ggaagttcac
     3481 gccggtgaac ttggacttgt ggtagaaggt gttgcccctc aggctgatgt gggcgctcac
     3541 ggaggcggag ccgccgtcct cgaactccag ggacctctcc caggacaggc cctcaggaaa
     3601 ggcctgcttg aaatagtctg ggatctcttc tgggtacttg gtaaaaactc tgtggccgta
     3661 acagaacacg tgggacacca ggtcgaaggc gaaaggcatt ggcccgccct cggccattgt
     3721 gacgcgcagt gtcatctcct gatgtccctc gtaaggtctg cctgtgccct caccctcgat
     3781 tgtgaactca tgcccattga cggagccgtc catgtagtac ctcatcttca tctcgggctt
     3841 gatcacgctc accatggtag atccccctgg ggagagaggt cggtgattcg gtcaacgagg
     3901 gagccgactg ccgacgtgcg ctccggaggc ttgcagaatg cggaacaccg cgcgggcagg
     3961 aacagggccc acactaccgc cccacacccc gcctcccgca ccgccccttc ccggccgctg
     4021 ctctcggcgc gccctgctga gcagccgcta ttggccacag cccatcgcgg tcggcgcgct
     4081 gccattgctc cctggcgctg tccgtctgcg agggtactag tgagacgtgc ggcttccgtt
     4141 tgtcacgtcc ggcacgccgc gaaccgcaag gaaccttccc gacttagggg cggagcagga
     4201 agcgtcgccg gggggcccac aagggtagcg gcgaagatcc gggtgacgct gcgaacggac
     4261 gtgaagaatg tgcgagaccc agggtcggcg ccgctgcgtt tcccggaacc acgcccagag
     4321 cagccgcgtc cctgcgcaaa cccagggctg ccttggaaaa ggcgcaaccc caaccccctc
     4381 gagccgcggc caaagtggat ctctgctgtc cctgtaataa acccgaaaat tttgaatttt
     4441 tgtaatttgt ttttgtaatt ctttagtttg tatgtctgtt gctattatgt ctactattct
     4501 ttcccctgca ctgtaccccc caatcccccc ttttctttta aaattgtgga tgaatactgc
     4561 catttgtctc gaggtcgaga attcaaaaat gagacgacga aaggcatgta ctcgagtaca
     4621 tgcctttcgt cgtctcaccg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat
     4681 cgaaatactt tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa
     4741 ctgcaaacta cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg
     4801 tgtttacagt caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat
     4861 atgaaggaat catgggaaat aggccctcgg tgaagggggc ggccgctcga ggctagtctc
     4921 gtgatcgata ccgtcgagat ccgttcacta atcgaatgga tctgtctctg tctctctctc
     4981 caccttcttc ttctattcct tcgggcctgt cgggtcccct cggggttggg aggtgggtct
     5041 gaaacgataa tggtgaatat ccctgcctaa ctctattcac tatagaaagt acagcaaaaa
     5101 ctattcttaa acctaccaag cctcctacta tcattatgaa taattttata taccacagcc
     5161 aatttgttat gttaaaccaa ttccacaaac ttgcccattt atctaattcc aataattctt
     5221 gttcattctt ttcttgctgg ttttgcgatt cttcaattaa ggagtgtatt aagcttgtgt
     5281 aattgttaat ttctctgtcc cactccatcc aggtcgtgtg attccaaatc tgttccagag
     5341 atttattact ccaactagca ttccaaggca cagcagtggt gcaaatgagt tttccagagc
     5401 aaccccaaat ccccaggagc tgttgatcct ttaggtatct ttccacagcc aggattcttg
     5461 cctggagctg cttgatgccc cagactgtga gttgcaacag atgctgttgc gcctcaatag
     5521 ccctcagcaa attgttctgc tgctgcacta taccagacaa taattgtctg gcctgtaccg
     5581 tcagcgtcat tgacgctgcg cccatagtgc ttcctgctgc tcccaagaac ccaaggaaca
     5641 aagctcctat tcccactgct cttttttctc tctgcaccac tcttctcttt gccttggtgg
     5701 gtgctactcc taatggttca atttttacta ctttatattt atataattca cttctccaat
     5761 tgtccctcat atctcctcct ccaggtctga agatcagcgg ccgcttgctg tgcggtggtc
     5821 ttacttttgt tttgctcttc ctctatcttg tctaaagctt ccttggtgtc ttttatctct
     5881 atcctttgat gcacacaata gagggttgct actgtattat ataatgatct aagttcttct
     5941 gatcctgtct gaagggatgg ttgtagctgt cccagtattt gtctacagcc ttctgatgtt
     6001 tctaacaggc caggattaac tgcgaatcgt tctagctccc tgcttgccca tactatatgt
     6061 tttaatttat attttttctt tccccctggc cttaaccgaa ttttttccca tcgcgatcta
     6121 attctccccc gcttaatact gacgctctcg cacccatctc tctccttcta gcctccgcta
     6181 gtcaaaattt ttggcgtact caccagtcgc cgcccctcgc ctcttgccgt gcgcgcttca
     6241 gcaagccgag tcctgcgtcg agagagctcc tctggtttcc ctttcgcttt caagtccctg
     6301 ttcgggcgcc actgctagag attttccaca ctgactaaaa gggtctgagg gatctctagt
     6361 taccagagtc acacaacaga cgggcacaca ctacttgaag cactcaaggc aagctttatt
     6421 gaggcttaag cagtgggttc cctagttagc cagagagctc ccaggctcag atctggtcta
     6481 accagagaga cccgtttatg tatcgagcta ggcacttaaa tacaatatct ctgcaatgcg
     6541 gcaattcagt ggttcgtcca atccatgtca gacccgtctg ttgccttcct aataaggcac
     6601 gatcgtacca ccttacttcc accaatcggc atgcacggtg ctttttctct ccttgtaagg
     6661 catgttgcta actcatcgtt accatgttgc aagactacaa gagtattgca taagactaca
     6721 ttaagcttgc agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     6781 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     6841 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     6901 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     6961 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     7021 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     7081 ggcggtaata cggttatcca ca
//
