LOCUS       sequence-376525-        7539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd gen lentiviral backbone for cloning and expression of new shRNA 
            sequences. Uses hygromycin for selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7539)
  TITLE     empty vector
REFERENCE   2  (bases 1 to 7539)
  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"
COMMENT     Sequence Label: pLKO.1 hygro
FEATURES             Location/Qualifiers
     source          1..7539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     375..394
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     628..645
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    762..783
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(798..803,804..821,822..828)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    836..852
                     /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     841..863
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     860..876
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     860..876
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     895..915
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        897..915
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        943..1169
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1170..1350
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1397..1522
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2015..2248
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2433..2477
                     /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        2775..3023
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2775..2795
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2946..2965
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    3086..3203
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3252..3762
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3681..3699
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3852..4877
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3852..4877
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(4901..4917)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4902..4918)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5000..5233
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5305..5426
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5342..5361)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5396..5415
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5466..5601
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5621..5640)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5622..5640)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5650..5667)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5650..5666)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5659..5681)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5808..6263
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5895..5914)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6105..6126
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6289..6393
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6394..6462,6463..7254)
                     /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(6612..6631)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7425..7539,1..474)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
       61 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
      121 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
      181 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
      241 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
      301 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
      361 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
      421 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
      481 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
      541 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
      601 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
      661 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
      721 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
      781 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
      841 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
      901 aaccctcact aaagggaaca aaagctggag ctgcaagctt aatgtagtct tatgcaatac
      961 tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa
     1021 agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc
     1081 aacagacggg tctgacatgg attggacgaa ccactgaatt gccgcattgc agagatattg
     1141 tatttaagtg cctagctcga tacataaacg ggtctctctg gttagaccag atctgagcct
     1201 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     1261 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     1321 ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa
     1381 agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag
     1441 aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga
     1501 gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa
     1561 aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag
     1621 cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag
     1681 acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt
     1741 atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa
     1801 ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc
     1861 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
     1921 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
     1981 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
     2041 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
     2101 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
     2161 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
     2221 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
     2281 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
     2341 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
     2401 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
     2461 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
     2521 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
     2581 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc
     2641 cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca
     2701 gatccattcg attagtgaac ggatctcgac ggtatcgatc acgagactag cctcgagcgg
     2761 ccgccccctt caccgagggc ctatttccca tgattccttc atatttgcat atacgataca
     2821 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     2881 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     2941 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     3001 tcttgtggaa aggacgaaac accggtccgc aggtatgcac gcgtgaattc tcgacctcga
     3061 gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
     3121 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
     3181 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc
     3241 cgcggctcga gggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga
     3301 cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca
     3361 ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg
     3421 gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg
     3481 acgtgacaaa cggaagccgc acgtctcact agtaccctcg cagacggaca gcgccaggga
     3541 gcaatggcag cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc
     3601 gccgagagca gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg
     3661 gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca
     3721 gtcggctccc tcgttgaccg aatcaccgac ctctctcccc agggggatcc accggatcaa
     3781 gcttgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcagatct gatcccgggg
     3841 ggcaatgaga tatgaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     3901 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     3961 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     4021 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     4081 tgcttgacat tggggaattt agcgagagcc tgacctattg catctcccgc cgtgcacagg
     4141 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     4201 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     4261 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     4321 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     4381 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     4441 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     4501 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     4561 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     4621 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     4681 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     4741 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     4801 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     4861 cgagggcaaa ggaatagagt agatgccgac cgggatctat cgataccgtc gacctcgagg
     4921 gggggcccgg tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     4981 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     5041 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     5101 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     5161 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     5221 aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     5281 aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa
     5341 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5401 ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc
     5461 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     5521 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     5581 gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta
     5641 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     5701 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     5761 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     5821 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5881 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5941 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     6001 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     6061 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     6121 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     6181 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     6241 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     6301 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     6361 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6421 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6481 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6541 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6601 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6661 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6721 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6781 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6841 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6901 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6961 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     7021 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     7081 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     7141 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     7201 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     7261 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     7321 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     7381 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7441 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7501 ccggatcaag agctaccaac tctttttccg aaggtaact
//
