LOCUS       sequence-128044-        6340 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral vector for shRNA expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6340)
  AUTHORS   Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, 
            Sabatini DM, Chen IS, Hahn WC, Sharp PA, Weinberg RA, Novina CD
  TITLE     Lentivirus-delivered stable gene silencing by RNAi in primary cells.
  JOURNAL   RNA 2003 Apr;9(4):493-501.
  PUBMED    12649500
REFERENCE   2  (bases 1 to 6340)
  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: "RNA"; date:
            "2003-04"; volume: "9(4)"; pages: "493-501"
COMMENT     Sequence Label: pMKO.1 puro
FEATURES             Location/Qualifiers
     source          1..6340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        310..639
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      490..625
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             649..1248
                     /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(649..668)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1145..1165
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1444..1869
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(1972..1994)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2135..2464
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2315..2450
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2486..2505
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2586..2603)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2757..3345)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2837..2856)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3516..4307,4308..4376))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4139..4158
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4377..4481)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4549..4567
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        4667..5046
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5047..5250
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5201..5221
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5251..5426
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    5489..5688
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             5889..6305
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6235..6257
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6273..6289
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gtacgcgtac tagtactccc atatggaatt cgccagcaca gtggtcgacc
      301 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gcttcccagc aggcagaagt
      361 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
      421 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
      481 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
      541 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
      601 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agcttaccat gaccgagtac
      661 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
      721 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
      781 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
      841 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
      901 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
      961 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     1021 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     1081 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     1141 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     1201 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac
     1261 gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcgattag tccaatttgt
     1321 taaagacagg atatcagtgg tccaggctct agttttgact caacaatatc accagctgaa
     1381 gcctatagag tacgagccat agataaaata aaagatttta tttagtctcc agaaaaaggg
     1441 gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa
     1501 ggcatggaaa aatacataac tgagaataga gaagttcaga tcaaggtcag gaacagatgg
     1561 aacagggtcg accctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg
     1621 accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc
     1681 tgctccccga gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat
     1741 tgactgagtc gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt
     1801 gtggtctcgc tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg
     1861 tctttcattt gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac
     1921 caccgggagg taagctggct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     1981 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     2041 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     2101 tagcgatagc ggagtgtaga tccggctgtg gaatgtgtgt cagttagggt gtggaaagtc
     2161 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     2221 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     2281 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     2341 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     2401 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     2461 caaaaagctt actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     2521 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     2581 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2641 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2701 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2761 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2821 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2881 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2941 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3001 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3061 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3121 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3181 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3241 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3301 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3361 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3421 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3481 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3541 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3601 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3661 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3721 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3781 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc
     3841 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3901 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3961 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4021 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4081 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat
     4141 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4201 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4261 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4321 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4381 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4441 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4501 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     4561 acgaggccct ttcgtcttca agaattagct tggccattgc atacgttgta tccatatcat
     4621 aatatgtaca tttatattgg ctcatgtcca acattaccgc catgttgaca ttgattattg
     4681 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     4741 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     4801 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     4861 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     4921 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     4981 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     5041 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     5101 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     5161 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     5221 gtacggtggg aggtctatat aagcagagct caataaaaga gcccacaacc cctcactcgg
     5281 cgcgccagtc ttccgataga ctgcgtcgcc cgggtacccg tattcccaat aaagcctctt
     5341 gctgtttgca tccgaatcgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg
     5401 actacccacg acgggggtct ttcatttggg ggctcgtccg ggatttggag acccctgccc
     5461 agggaccacc gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg
     5521 attgtctagt gtctatgttt gatgttatgc gcctgcgtct gtactagtta gctaactagc
     5581 tctgtatctg gcggacccgt ggtggaactg acgagttctg aacacccggc cgcaaccctg
     5641 ggagacgtcc cagggacttt gggggccgtt tttgtggccc gacctgagga agggagtcga
     5701 tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg agacgagaac ctaaaacagt
     5761 tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg aagccgcgcg tcttgtctgc
     5821 tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac tgtgtttctg tatttgtctg
     5881 aaaattaggg ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg
     5941 tcgagcggat cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct
     6001 gctctgcaga atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag
     6061 acctcatcac ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc
     6121 aggtccccta catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc
     6181 cctttgtaca ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg
     6241 aacctcctcg ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag
     6301 gcgccggaat tgaagatctg tcgacaaggt cgggcaggaa
//
