LOCUS       Exported                8919 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Backbone for lentiviral gene silencing with monomeric 
            Kusabira-Orange2 expression.
ACCESSION   .
VERSION     .
KEYWORDS    pLKO.1-TRC.mKO2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8919)
  AUTHORS   Dias
  TITLE     Transcriptomic data of triple negative breast cancer revealed GBP1 
            as potential new therapeutic target
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8919)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8919
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(101..689)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(181..200)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(860..1720)
                     /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     1483..1502
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1721..1825)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1851..2306)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1988..2009)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2200..2219
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2433..2455
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2447..2464
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2448..2464
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2474..2493
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2474..2492
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2513..2648
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2567..2586)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2688..2809
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2699..2718)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2753..2772
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             2881..3114
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             complement(3216..3869)
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        complement(3881..4391)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3944..3962)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    4440..4557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(6479..6719)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     complement(6529..6548)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(6699..6719)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(7017..7061)
                     /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    7246..7479
                     /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    7972..8097
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             8144..8324
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(8325..8551)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(8579..8599)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(8579..8597)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8618..8634)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8618..8634)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8631..8653)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8642..8658
                     /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(8666..8696)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8711..8732
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8849..8866)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
       61 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
      121 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
      181 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
      241 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
      301 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
      361 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
      421 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
      481 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
      541 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
      601 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
      661 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
      721 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
      781 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
      841 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
      901 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
      961 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     1021 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     1081 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     1141 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     1201 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     1261 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     1321 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     1381 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     1441 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     1501 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     1561 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     1621 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     1681 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     1741 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     1801 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     1861 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     1921 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     1981 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     2041 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     2101 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     2161 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     2221 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     2281 gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga
     2341 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     2401 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     2461 cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtacgtccc taggcctcca
     2521 aaaaagcctc ctcactactt ctggaatagc tcagaggccg aggcggcctc ggcctctgca
     2581 taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg
     2641 ggcgggatgg gcggagttag gggcgggata gctagagcca gacatgataa gatacattga
     2701 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     2761 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttc ctctcactct
     2821 ctgatattca tttctttgca agttataaat actgaataat aagatgacat gaactactac
     2881 tgctagagat tttccacact gactaaaagg gtctgaggga tctctagtta ccagagtcac
     2941 acaacagacg ggcacacact acttgaagca ctcaaggcaa gctttattga ggcttaagca
     3001 gtgggttccc tagttagcca gagagctccc aggctcagat ctggtctaac cagagagacc
     3061 cagtacaagc aaaaagcaga tcttgtcttc gttgggagtg aattagccct tccagtcccc
     3121 ccttttcttt taaaaagtgg ctaagatcta cagctgcctt gtaagtcatt ggtcttaaag
     3181 gtacccgacc cacctccgcc cgagcctccg ccaccggagt gggccacggc gtcctccacc
     3241 tgctcggtga tgttgccctc ggtcttcctc accagcctgt ggccgatgta gtggtcgccg
     3301 ggcatctcca ggatctcctt ggcggccttg taggtggtct tcatctggca cttgtggttg
     3361 ccgccgccct ccagcttcag gtacatggtc acgtcgccct tcagcacgcc gtcgctggcg
     3421 gtgatcttct cggtggaggg ctcccagtcc acgctctggt tctgcatgat ggggccgtcg
     3481 gcggggaagt tcacgccggt gaacttggac ttgtggtaga aggtgttgcc cctcaggctg
     3541 atgtgggcgc tcacggaggc ggagccgccg tcctcgaact ccagggacct ctcccaggac
     3601 aggccctcag gaaaggcctg cttgaaatag tctgggatct cttctgggta cttggtaaaa
     3661 actctgtggc cgtaacagaa cacgtgggac accaggtcga aggcgaaagg cattggcccg
     3721 ccctcggcca ttgtgacgcg cagtgtcatc tcctgatgtc cctcgtaagg tctgcctgtg
     3781 ccctcaccct cgattgtgaa ctcatgccca ttgacggagc cgtccatgta gtacctcatc
     3841 ttcatctcgg gcttgatcac gctcaccatg gtagatcccc ctggggagag aggtcggtga
     3901 ttcggtcaac gagggagccg actgccgacg tgcgctccgg aggcttgcag aatgcggaac
     3961 accgcgcggg caggaacagg gcccacacta ccgccccaca ccccgcctcc cgcaccgccc
     4021 cttcccggcc gctgctctcg gcgcgccctg ctgagcagcc gctattggcc acagcccatc
     4081 gcggtcggcg cgctgccatt gctccctggc gctgtccgtc tgcgagggta ctagtgagac
     4141 gtgcggcttc cgtttgtcac gtccggcacg ccgcgaaccg caaggaacct tcccgactta
     4201 ggggcggagc aggaagcgtc gccggggggc ccacaagggt agcggcgaag atccgggtga
     4261 cgctgcgaac ggacgtgaag aatgtgcgag acccagggtc ggcgccgctg cgtttcccgg
     4321 aaccacgccc agagcagccg cgtccctgcg caaacccagg gctgccttgg aaaaggcgca
     4381 accccaaccc cctcgagccg cggccaaagt ggatctctgc tgtccctgta ataaacccga
     4441 aaattttgaa tttttgtaat ttgtttttgt aattctttag tttgtatgtc tgttgctatt
     4501 atgtctacta ttctttcccc tgcactgtac cccccaatcc ccccttttct tttaaaattg
     4561 tggatgaata ctgccatttg tctcgaggtc gagaattcta caaaaaagag caagaagcta
     4621 aaaaagattt aaaaattatt tttagcgcag ttaatggaac aggaactaaa tttaccccaa
     4681 aaatattacg tgaatcagga tataacgtta ttgaggttga agagcatgca tttgaagatg
     4741 aaacatttaa aaatgttgta aatccaaatc cagaatttga tcctgcatga aaaataccgc
     4801 ttgaatatgg tattaaacat gatgcagata ttattattat gaatgaccca gatgctgaca
     4861 gatttggaat ggcaataaaa catgatggtc attttgtaag attagatgga aatcaaacag
     4921 gaccaatttt aattgattga aaattatcaa atctaaaacg cttaaatagc attccaaaaa
     4981 atccggctct atattcaagt tttgtaacaa gtgatttggg tgatagaatc gctcatgaaa
     5041 aatatggagt taatattgta aaaactttaa ctggatttaa atgaatgggt agagaaattg
     5101 ctaaagaaga agataacgga ttaaattttg tttttgctta tgaagaaagt tatggatatg
     5161 taattgatga ctcagctaga gataaagatg gaatacaagc ttctatatta atagcagagg
     5221 ctgcttgatt ttataaaaaa caaaataaaa cattagtaga ctatttagaa gatttattta
     5281 aagaaatggg tgcatattac actttcactt taaacttgaa ttttaaacca gaagaaaaga
     5341 aattaaaaat tgaaccatta atgaaatcat tgagagcaac acccttaact caaattgctg
     5401 gacttaaagt tgttaatgtt gaagactaca tcgatggaat gtataatatg ccaggacaag
     5461 acttactaaa attttattta gaagataagt catgatttgc tgttcgccca agtggaactg
     5521 aacctaaact aaaaatttat tttataggtg ttggtgaatc tgttcaaaac gctaaagtta
     5581 aagtagacga aattattaaa gaattaaaat taaaaatgaa tatataggag aaaaaatgaa
     5641 actaaacaaa tatatagatc acacattatt aaaacaagat gctacgaaag ctgaaattaa
     5701 acaattatgt gatgaagcaa ttgaatttga ttttgcaaca gtttgtgtta attcatattg
     5761 aacaagctat tgtaaagaat tattaaaagg cacaaatgta ggaataacaa atgttgtagg
     5821 ttttcctcta ggtgcatgca caacagctac aaaagcattc gaagtttctg aagcaattaa
     5881 agatggtgca acagaaattg atatggtatt aaatattggt gcattaaaag acaaaaatta
     5941 tgaattagtt ttagaagaca tgaaagctgt aaaaaaagca gctggatcac atgttgttaa
     6001 atgtattatg gaaaattgtt tattaacaaa agaagaaatc atgaaagctt gtgaaatagc
     6061 tgttgaagct ggattagaat ttgttaaaac atcaacagga ttttcaaaat caggtgcaac
     6121 atttgaagat gttaaactaa tgaagtcagt tgttaaagac aatgctttag ttaaagcagc
     6181 tggtggagtt agaacatttg aagatgctca aaaaatgatt gaagcaggag ctgaccgctt
     6241 aggaacaagt ggtggagtag ctattattaa aggtgaagaa aacaacgcga gttactaaaa
     6301 ctagcgtttt tttattttgc tcatttttat taaaagtttg caaaaaggaa cataaaaatt
     6361 ctaattattg atactaaagt tattaaaaag aagattttgg ttgattttat aaaggtcata
     6421 gaatataata ttttagcatg tgtattttgt gtgctcattt acaacacacc ggtgtttcgt
     6481 cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac ggtaagcata
     6541 tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa ttattacttt
     6601 ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat tccaattatc
     6661 tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa taggccctcg
     6721 gtgaaggggg cggccgctcg aggctagtct cgtgatcgat accgtcgaga tccgttcact
     6781 aatcgaatgg atctgtctct gtctctctct ccaccttctt cttctattcc ttcgggcctg
     6841 tcgggtcccc tcggggttgg gaggtgggtc tgaaacgata atggtgaata tccctgccta
     6901 actctattca ctatagaaag tacagcaaaa actattctta aacctaccaa gcctcctact
     6961 atcattatga ataattttat ataccacagc caatttgtta tgttaaacca attccacaaa
     7021 cttgcccatt tatctaattc caataattct tgttcattct tttcttgctg gttttgcgat
     7081 tcttcaatta aggagtgtat taagcttgtg taattgttaa tttctctgtc ccactccatc
     7141 caggtcgtgt gattccaaat ctgttccaga gatttattac tccaactagc attccaaggc
     7201 acagcagtgg tgcaaatgag ttttccagag caaccccaaa tccccaggag ctgttgatcc
     7261 tttaggtatc tttccacagc caggattctt gcctggagct gcttgatgcc ccagactgtg
     7321 agttgcaaca gatgctgttg cgcctcaata gccctcagca aattgttctg ctgctgcact
     7381 ataccagaca ataattgtct ggcctgtacc gtcagcgtca ttgacgctgc gcccatagtg
     7441 cttcctgctg ctcccaagaa cccaaggaac aaagctccta ttcccactgc tcttttttct
     7501 ctctgcacca ctcttctctt tgccttggtg ggtgctactc ctaatggttc aatttttact
     7561 actttatatt tatataattc acttctccaa ttgtccctca tatctcctcc tccaggtctg
     7621 aagatcagcg gccgcttgct gtgcggtggt cttacttttg ttttgctctt cctctatctt
     7681 gtctaaagct tccttggtgt cttttatctc tatcctttga tgcacacaat agagggttgc
     7741 tactgtatta tataatgatc taagttcttc tgatcctgtc tgaagggatg gttgtagctg
     7801 tcccagtatt tgtctacagc cttctgatgt ttctaacagg ccaggattaa ctgcgaatcg
     7861 ttctagctcc ctgcttgccc atactatatg ttttaattta tattttttct ttccccctgg
     7921 ccttaaccga attttttccc atcgcgatct aattctcccc cgcttaatac tgacgctctc
     7981 gcacccatct ctctccttct agcctccgct agtcaaaatt tttggcgtac tcaccagtcg
     8041 ccgcccctcg cctcttgccg tgcgcgcttc agcaagccga gtcctgcgtc gagagagctc
     8101 ctctggtttc cctttcgctt tcaagtccct gttcgggcgc cactgctaga gattttccac
     8161 actgactaaa agggtctgag ggatctctag ttaccagagt cacacaacag acgggcacac
     8221 actacttgaa gcactcaagg caagctttat tgaggcttaa gcagtgggtt ccctagttag
     8281 ccagagagct cccaggctca gatctggtct aaccagagag acccgtttat gtatcgagct
     8341 aggcacttaa atacaatatc tctgcaatgc ggcaattcag tggttcgtcc aatccatgtc
     8401 agacccgtct gttgccttcc taataaggca cgatcgtacc accttacttc caccaatcgg
     8461 catgcacggt gctttttctc tccttgtaag gcatgttgct aactcatcgt taccatgttg
     8521 caagactaca agagtattgc ataagactac attaagcttg cagctccagc ttttgttccc
     8581 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     8641 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     8701 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     8761 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     8821 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     8881 ggctgcggcg agcggtatca gctcactcaa aggcggtaa
//
