LOCUS       sequence-218859-        7101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TRCN0000048179 (Target GTCCCTGCATATCGTCATGTT), silence human ARL4C 
            gene 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-TRC.mKO2_shARL4C.1
FEATURES             Location/Qualifiers
     source          1..7101
                     /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    2177..2294
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2343..2853
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2772..2790
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2865..3518
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             2865..3518
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     LTR             3620..3853
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3925..4046
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3962..3981)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4016..4035
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4086..4221
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4241..4260)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4242..4260)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4270..4287)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4270..4286)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4279..4301)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4428..4883
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4515..4534)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4725..4746
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4909..5013
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5014..5082,5083..5874)
                     /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(5232..5251)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6045..6633
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6534..6553
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6787..6804
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6921..6942
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6957..6962,6963..6980,6981..6987)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6995..7011
                     /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     7000..7022
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7019..7035
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7019..7035
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7054..7074
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7056..7074
                     /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 cgggtccctg catatcgtca
     2101 tgttctcgag aacatgacga tatgcaggga ctttttgatt ctcgacctcg agacaaatgg
     2161 cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa
     2221 gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa
     2281 aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg ccgcggctcg
     2341 agggggttgg ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc
     2401 tctgggcgtg gttccgggaa acgcagcggc gccgaccctg ggtctcgcac attcttcacg
     2461 tccgttcgca gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt
     2521 cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa
     2581 acggaagccg cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca
     2641 gcgcgccgac cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc
     2701 agcggccggg aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc
     2761 ctgcccgcgc ggtgttccgc attctgcaag cctccggagc gcacgtcggc agtcggctcc
     2821 ctcgttgacc gaatcaccga cctctctccc cagggggatc taccatggtg agcgtgatca
     2881 agcccgagat gaagatgagg tactacatgg acggctccgt caatgggcat gagttcacaa
     2941 tcgagggtga gggcacaggc agaccttacg agggacatca ggagatgaca ctgcgcgtca
     3001 caatggccga gggcgggcca atgcctttcg ccttcgacct ggtgtcccac gtgttctgtt
     3061 acggccacag agtttttacc aagtacccag aagagatccc agactatttc aagcaggcct
     3121 ttcctgaggg cctgtcctgg gagaggtccc tggagttcga ggacggcggc tccgcctccg
     3181 tgagcgccca catcagcctg aggggcaaca ccttctacca caagtccaag ttcaccggcg
     3241 tgaacttccc cgccgacggc cccatcatgc agaaccagag cgtggactgg gagccctcca
     3301 ccgagaagat caccgccagc gacggcgtgc tgaagggcga cgtgaccatg tacctgaagc
     3361 tggagggcgg cggcaaccac aagtgccaga tgaagaccac ctacaaggcc gccaaggaga
     3421 tcctggagat gcccggcgac cactacatcg gccacaggct ggtgaggaag accgagggca
     3481 acatcaccga gcaggtggag gacgccgtgg cccactccgg tggcggaggc tcgggcggag
     3541 gtgggtcggg tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     3601 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     3661 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     3721 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     3781 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     3841 aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     3901 aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa
     3961 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4021 ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc
     4081 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4141 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     4201 gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta
     4261 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     4321 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     4381 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     4441 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4501 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4561 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4621 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     4681 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4741 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     4801 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4861 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     4921 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4981 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5041 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5101 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5161 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5221 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5281 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5341 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5401 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5461 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5521 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5581 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5641 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5701 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5761 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5821 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5881 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5941 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6001 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6061 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6121 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6181 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6241 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6301 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6361 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6421 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6481 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6541 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6601 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6661 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6721 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     6781 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     6841 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     6901 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     6961 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     7021 ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa
     7081 aagctggagc tgcaagctta a
//
