LOCUS       Exported                8102 bp ds-DNA     circular SYN 19-JAN-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pLKO.3G
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8102)
  TITLE     pLKO variants
REFERENCE   2  (bases 1 to 8102)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 19, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8102
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     165..184
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     424..445
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      487..496
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        complement(512..1022)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(575..593)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    1071..1188
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(1288..1528)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     complement(1338..1357)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1508..1528)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(2045..2089)
                     /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    2274..2507
                     /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    3000..3125
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             3172..3352
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(3353..3579)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(3607..3627)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3607..3625)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3646..3662)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3646..3662)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3659..3681)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3670..3686
                     /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(3694..3724)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3739..3760
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3877..3894)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4048..4636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4128..4147)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4807..5667)
                     /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     5430..5449
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5668..5772)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5798..6253)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5935..5956)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6147..6166
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6380..6402
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6394..6411
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6395..6411
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6421..6440
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6421..6439
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      6460..6595
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6514..6533)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    6635..6756
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6646..6665)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6700..6719
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             6828..7061
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     7246..7262
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     7247..7263
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    7266..7854
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             7372..7383
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7781..7801
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7873..490)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(7876..490)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7918..7939)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt
       61 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
      121 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
      181 cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
      241 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg
      301 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg
      361 tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt
      421 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
      481 tgctcaccat ggtggcgacc ggtggatccc cctggggaga gaggtcggtg attcggtcaa
      541 cgagggagcc gactgccgac gtgcgctccg gaggcttgca gaatgcggaa caccgcgcgg
      601 gcaggaacag ggcccacact accgccccac accccgcctc ccgcaccgcc ccttcccggc
      661 cgctgctctc ggcgcgccct gctgagcagc cgctattggc cacagcccat cgcggtcggc
      721 gcgctgccat tgctccctgg cgctgtccgt ctgcgagggt actagtgaga cgtgcggctt
      781 ccgtttgtca cgtccggcac gccgcgaacc gcaaggaacc ttcccgactt aggggcggag
      841 caggaagcgt cgccgggggg cccacaaggg tagcggcgaa gatccgggtg acgctgcgaa
      901 cggacgtgaa gaatgtgcga gacccagggt cggcgccgct gcgtttcccg gaaccacgcc
      961 cagagcagcc gcgtccctgc gcaaacccag ggctgccttg gaaaaggcgc aaccccaacc
     1021 ccctcgagcc gcggccaaag tggatctctg ctgtccctgt aataaacccg aaaattttga
     1081 atttttgtaa tttgtttttg taattcttta gtttgtatgt ctgttgctat tatgtctact
     1141 attctttccc ctgcactgta ccccccaatc cccccttttc ttttaaaatt gtggatgaat
     1201 actgccattt gtctcgaggt cgaaaactgg tttaattaag tagctcgagg tggaattcac
     1261 gcaagcatac ctgcggaccg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat
     1321 cgaaatactt tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa
     1381 ctgcaaacta cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg
     1441 tgtttacagt caaattaatt ctaattatct ctctaacagc cttgtatcgt atatgcaaat
     1501 atgaaggaat catgggaaat aggccctctt cctgcccgac cttgtcgaca ggactcgact
     1561 gacgcgtgta cccccttact tgaggataaa ttatgtctaa tattcaaact ggcgccgagc
     1621 gtatgccgca tgacctttcc catcttggct tccttgctgg tcagattggt cgtcttatta
     1681 ccatttcaac tactccggtt atcgctggcg actccttcga gatggacgcc gttggcgctc
     1741 tccgtctttc tccattgcgt cgtggtcgag gctagtctcg tgatcgatac cgtcgagatc
     1801 cgttcactaa tcgaatggat ctgtctctgt ctctctctcc accttcttct tctattcctt
     1861 cgggcctgtc gggtcccctc ggggttggga ggtgggtctg aaacgataat ggtgaatatc
     1921 cctgcctaac tctattcact atagaaagta cagcaaaaac tattcttaaa cctaccaagc
     1981 ctcctactat cattatgaat aattttatat accacagcca atttgttatg ttaaaccaat
     2041 tccacaaact tgcccattta tctaattcca ataattcttg ttcattcttt tcttgctggt
     2101 tttgcgattc ttcaattaag gagtgtatta agcttgtgta attgttaatt tctctgtccc
     2161 actccatcca ggtcgtgtga ttccaaatct gttccagaga tttattactc caactagcat
     2221 tccaaggcac agcagtggtg caaatgagtt ttccagagca accccaaatc cccaggagct
     2281 gttgatcctt taggtatctt tccacagcca ggattcttgc ctggagctgc ttgatgcccc
     2341 agactgtgag ttgcaacaga tgctgttgcg cctcaatagc cctcagcaaa ttgttctgct
     2401 gctgcactat accagacaat aattgtctgg cctgtaccgt cagcgtcatt gacgctgcgc
     2461 ccatagtgct tcctgctgct cccaagaacc caaggaacaa agctcctatt cccactgctc
     2521 ttttttctct ctgcaccact cttctctttg ccttggtggg tgctactcct aatggttcaa
     2581 tttttactac tttatattta tataattcac ttctccaatt gtccctcata tctcctcctc
     2641 caggtctgaa gatcagcggc cgcttgctgt gcggtggtct tacttttgtt ttgctcttcc
     2701 tctatcttgt ctaaagcttc cttggtgtct tttatctcta tcctttgatg cacacaatag
     2761 agggttgcta ctgtattata taatgatcta agttcttctg atcctgtctg aagggatggt
     2821 tgtagctgtc ccagtatttg tctacagcct tctgatgttt ctaacaggcc aggattaact
     2881 gcgaatcgtt ctagctccct gcttgcccat actatatgtt ttaatttata ttttttcttt
     2941 ccccctggcc ttaaccgaat tttttcccat cgcgatctaa ttctcccccg cttaatactg
     3001 acgctctcgc acccatctct ctccttctag cctccgctag tcaaaatttt tggcgtactc
     3061 accagtcgcc gcccctcgcc tcttgccgtg cgcgcttcag caagccgagt cctgcgtcga
     3121 gagagctcct ctggtttccc tttcgctttc aagtccctgt tcgggcgcca ctgctagaga
     3181 ttttccacac tgactaaaag ggtctgaggg atctctagtt accagagtca cacaacagac
     3241 gggcacacac tacttgaagc actcaaggca agctttattg aggcttaagc agtgggttcc
     3301 ctagttagcc agagagctcc caggctcaga tctggtctaa ccagagagac ccgtttatgt
     3361 atcgagctag gcacttaaat acaatatctc tgcaatgcgg caattcagtg gttcgtccaa
     3421 tccatgtcag acccgtctgt tgccttccta ataaggcacg atcgtaccac cttacttcca
     3481 ccaatcggca tgcacggtgc tttttctctc cttgtaaggc atgttgctaa ctcatcgtta
     3541 ccatgttgca agactacaag agtattgcat aagactacat taagcttgca gctccagctt
     3601 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     3661 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3721 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3781 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3841 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3901 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3961 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4021 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4081 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4141 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4201 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4261 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4321 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4381 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4441 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4501 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4561 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4621 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4681 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4741 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4801 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4861 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4921 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4981 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5041 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5101 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5161 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5221 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5281 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5341 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5401 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5461 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5521 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5581 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5641 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5701 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5761 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     5821 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     5881 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     5941 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     6001 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     6061 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     6121 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     6181 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     6241 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     6301 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     6361 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg
     6421 taatacgact cactataggg cgaattgggt acgtccctag gcctccaaaa aagcctcctc
     6481 actacttctg gaatagctca gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa
     6541 ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg
     6601 gagttagggg cgggatagct agagccagac atgataagat acattgatga gtttggacaa
     6661 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     6721 ttatttgtaa ccattataag ctgcaataaa caagttcctc tcactctctg atattcattt
     6781 ctttgcaagt tataaatact gaataataag atgacatgaa ctactactgc tagagatttt
     6841 ccacactgac taaaagggtc tgagggatct ctagttacca gagtcacaca acagacgggc
     6901 acacactact tgaagcactc aaggcaagct ttattgaggc ttaagcagtg ggttccctag
     6961 ttagccagag agctcccagg ctcagatctg gtctaaccag agagacccag tacaagcaaa
     7021 aagcagatct tgtcttcgtt gggagtgaat tagcccttcc agtcccccct tttcttttaa
     7081 aaagtggcta agatctacag ctgccttgta agtcattggt cttaaaggta cctgaggtgt
     7141 gactggaaaa cccacctcct cctcctcttg tgcttctagc caggcacaat cagcattggt
     7201 agctgctgta ttgctacttg tgattgctcc atgtttttct aggtctcgag gtcgacggta
     7261 tcgatgcggg gaggcggccc aaagggagat ccgactcgtc tgagggcgaa ggcgaagacg
     7321 cggaagaggc cgcagagccg gcagcaggcc gcgggaagga aggtccgctg gattgagggc
     7381 cgaagggacg tagcagaagg acgtcccgcg cagaatccag gtggcaacac aggcgagcag
     7441 ccaaggaaag gacgatgatt tccccgacaa caccacggaa ttgtcagtgc ccaacagccg
     7501 agcccctgtc cagcagcggg caaggcaggc ggcgatgagt tccgccgtgg caatagggag
     7561 ggggaaagcg aaagtcccgg aaaggagctg acaggtggtg gcaatgcccc aaccagtggg
     7621 ggttgcgtca gcaaacacag tgcacaccac gccacgttgc ctgacaacgg gccacaactc
     7681 ctcataaaga gacagcaacc aggatttata caaggaggag aaaatgaaag ccatacggga
     7741 agcaatagca tgatacaaag gcattaaagc agcgtatcca catagcgtaa aaggagcaac
     7801 atagttaaga ataccagtca atctttcaca aattttgtaa tccagaggtt gattatcgat
     7861 aagcttgata tcttacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac
     7921 gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg
     7981 ggtgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg
     8041 ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt
     8101 ca
//
