LOCUS       sequence-200856-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  AUTHORS   Michaud K, Solomon DA, Oermann E, Kim JS, Zhong WZ, Prados MD, Ozawa
            T, James CD, Waldman T
  TITLE     Pharmacologic inhibition of cyclin-dependent kinases 4 and 6 arrests
            the growth of glioblastoma multiforme intracranial xenografts.
  JOURNAL   Cancer Res. 2010 Apr 15. 70(8):3228-38.
  PUBMED    20354191
REFERENCE   2  (bases 1 to 7084)
  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: "Cancer Res.
            2010 Apr 15. 70(8):3228-38."
COMMENT     Sequence Label: pLKO-RB1-shRNA63
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    105..126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(141..146,147..164,165..171)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    179..195
                     /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     184..206
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     203..219
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     203..219
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     238..258
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        240..258
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        286..512
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             513..693
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    740..865
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1358..1591
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1776..1820
                     /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        2118..2366
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2118..2138
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2289..2308
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2463..2580
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2629..3139
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3058..3076
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3161..3760
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3161..3180)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3657..3677
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3888..4121
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4193..4314
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4230..4249)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4284..4303
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4354..4489
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4509..4528)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4510..4528)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4538..4555)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4538..4554)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4547..4569)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4696..5151
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4783..4802)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4993..5014
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5177..5281
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5282..5350,5351..6142)
                     /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(5500..5519)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6313..6901
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6802..6821
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7055..7072
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
       61 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
      121 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
      181 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
      241 attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa
      301 tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga
      361 aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa
      421 ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata
      481 ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag
      541 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
      601 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
      661 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
      721 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
      781 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
      841 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
      901 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
      961 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
     1021 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     1081 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     1141 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     1201 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     1261 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     1321 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     1381 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     1441 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     1501 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     1561 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     1621 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     1681 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     1741 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     1801 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     1861 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     1921 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
     1981 ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag
     2041 acagatccat tcgattagtg aacggatctc gacggtatcg atcacgagac tagcctcgag
     2101 cggccgcccc cttcaccgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     2161 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     2221 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     2281 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     2341 atatcttgtg gaaaggacga aacaccggcc acattatttc tagtccaaac tcgagtttgg
     2401 actagaaata atgtggtttt tgaattctcg acctcgagac aaatggcagt attcatccac
     2461 aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     2521 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     2581 cgggtttatt acagggacag cagagatcca ctttggccgc ggctcgaggg ggttggggtt
     2641 gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc
     2701 cgggaaacgc agcggcgccg accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt
     2761 cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct
     2821 aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg
     2881 tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg
     2941 atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg
     3001 ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg
     3061 ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat
     3121 caccgacctc tctccccagg gggatccacc ggagcttacc atgaccgagt acaagcccac
     3181 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     3241 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     3301 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     3361 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     3421 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     3481 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     3541 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     3601 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     3661 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     3721 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca
     3781 gcgcccgacc gaaaggagcg cacgacccca tgcatcggta cctttaagac caatgactta
     3841 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     3901 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca
     3961 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     4021 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     4081 atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct
     4141 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     4201 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4261 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4321 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4381 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4441 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac
     4501 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     4561 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     4621 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     4681 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4741 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4801 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4861 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4921 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4981 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5041 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5101 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     5161 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5221 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5281 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5341 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5401 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5461 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5521 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5581 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5641 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5701 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5761 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5821 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5881 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5941 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6001 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6061 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6121 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6181 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6241 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6301 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6361 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6421 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6481 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6541 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6601 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6661 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6721 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6781 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6841 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6901 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6961 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7021 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     7081 agag
//
