LOCUS       sequence-322047-        5560 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral vector for negative control of knockdown expressing a 
            non-target miR against Renilla luciferase and expression of a Ly6G 
            surface marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5560)
  AUTHORS   van der Donk LEH, van der Spek J, van Duivenvoorde T, Ten Brink MS, 
            Geijtenbeek TBH, Kuijl CP, van Heijst JWJ, Ates LS
  TITLE     An optimized retroviral toolbox for overexpression and genetic 
            perturbation of primary lymphocytes.
  JOURNAL   Biol Open. 2022 Feb 15;11(2):bio059032. doi: 10.1242/bio.059032. 
            Epub 2022 Mar 1.
  PUBMED    35229875
REFERENCE   2  (bases 1 to 5560)
  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: "Biol 
            Open."; date: "2022-02-15"; volume: "11(2):bio059032. doi"; pages: "
            10.1242/bio.059032. Epub 2022 Mar 1"
COMMENT     Sequence Label: pMY-Ly6G-Rluc_miR
FEATURES             Location/Qualifiers
     source          1..5560
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             22..615
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     CDS             1103..1519
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1487..1503
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1529..1907
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1845..1861
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      1968..1977
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     ncRNA           2414..2535
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2563..2577
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2634..2746
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     complement(3664..3681)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3835..4423)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3915..3934)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4587..5378,5379..5447))
                     /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     5210..5229
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5448..5552)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cccgaaaagt gccacctgca taatgaaaga ccccacctgt aggtttggca agctagctta
       61 agtaacgcca ttttgcaagg catggaaaaa tacataactg agaatagaaa agttcagatc
      121 aaggtcagga acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag
      181 ttcctgcccc ggctcagggc caagaacaga tggaacagct gaatatgggc caaacaggat
      241 atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc cccagatgcg
      301 gtccagccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc ccaaggacct
      361 gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct tctgttcgcg
      421 cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggc gcgccagtcc
      481 tccgattgac tgagtcgccc gggtacccgt attcccaata aagcctcttg ctgtttgcat
      541 ccgaatcgtg gactcgctga tccttgggag ggtctcctca gattgattga ctgcccacct
      601 cgggggtctt tcatttggag gttccaccga gatttggaga cccctgccca gggaccaccg
      661 acccccccgc cgggaggtaa gctggccagc ggtcgtttcg tgtctgtctc tgtctttgtg
      721 cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt actagttagc taactagctc
      781 tgtatctggc ggacccgtgg tggaactgac gagttcggaa cacccggccg caaccctggg
      841 agacgtccca gggacttcgg gggccgtttt tgtggcccga cctgagtcca aaaatcccga
      901 tcgttttgga ctctttggtg caccccccta ataggaggga tatgtggttc tggtaggaga
      961 cgagaaccta aaacagttcc cgcctccgtc tgaatttttg ctttcggttt gggaccgaag
     1021 ccgcgccgcg cgtcttgtct gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt
     1081 tctgtatttg tctgaaaatt agggccagac tgttaccact cccttaagtt tgaccttagg
     1141 tcactggaaa gatgtcgagc ggatcgctca caaccagtcg gtagatgtca agaagagacg
     1201 ttgggttacc ttctgctctg cagaatggcc aacctttaac gtcggatggc cgcgagacgg
     1261 cacctttaac cgagacctca tcacccaggt taagatcaag gtcttttcac ctggcccgca
     1321 tggacaccca gaccaggtcc cctacatcgt gacctgggaa gccttggctt ttgacccccc
     1381 tccctgggtc aagccctttg tacaccctaa gcctccgcct cctcttcctc catccgcccc
     1441 gtctctcccc cttgaacctc ctcgttcgac cccgcctcga tcctcccttt atccagccct
     1501 cactccttct ctaggcgccc ccatatggcc atatgagatc ttatatgggg cacccccgcc
     1561 ccttgtaaac ttccctgacc ctgacatgac aagagttact aacagcccct ctctccaagc
     1621 tcacttacag gctctctact tagtccagca cgaagtctgg agacctctgg cggcagccta
     1681 ccaagaacaa ctggaccgac cggccggtgg tacctcaccc ttaccgagtc ggcgacacag
     1741 tgtgggtccg ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag
     1801 tcctgctgac cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg
     1861 cccacgtgaa ggctgccgac cccgggggtg gaccatcctc tagactgccg gatctagcta
     1921 gttaattaac tccgtggatc cggtcgtgcg gccgcacgga aacgcgtgcc accatggaca
     1981 cctgtcatat cgccaagagc tgcgtgctga tcctgctggt ggttctgctg tgtgccgagc
     2041 gagcacaggg actggagtgc tacaactgta tcggcgtgcc acctgagaca agctgcaaca
     2101 ccaccacctg tcctttcagc gacggcttct gtgtggccct ggaaatcgaa gtgatcgtgg
     2161 acagccaccg cagcaaagtg aagtccaacc tgtgcctgcc tatctgcccc accacactgg
     2221 acaacaccga gatcacaggc aacgccgtga acgtgaaaac ctactgctgc aaagaggacc
     2281 tctgcaacgc cgctgttcca acaggcggaa gctcttggac aatggctggc gtgctgctgt
     2341 tcagcctggt gtctgttctg ctgcagacct tcctgtgagc atgccgcgac cctgcaggtt
     2401 tctgaagctt tgtttgaatg aggcttcagt actttacaga atcgttgcct gcacatcttg
     2461 gaaacacttg ctgggattac ttcttcaggt taacccaaca gaaggctcga gaaggtatat
     2521 tgctgttgac agtgagcgca ggaattataa tgcttatcta tagtgaagcc acagatgtat
     2581 agataagcat tataattcct atgcctactg cctcggactt caaggggcta gaattcaagg
     2641 ggctacttta ggagcaatta tcttgtttac taaaactgaa taccttgcta tctctttgat
     2701 acatttttac aaagctgaat taaaatggta taaattaaat cactttatcg attgcattgg
     2761 gtcgaccggg ccgccagcac agtggtcgat cgaggggggg cccgcgatta gtccaatttg
     2821 ttaaagacag gatatcagtg gtccaggctc tagttttgac tcaacaatat caccagctga
     2881 agcctataga gtacgagcca tagatagaat aaaagatttt atttagtctc cagaaaaagg
     2941 ggggaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca
     3001 aggcatggaa aatacataac tgagaataga gaagttcaga tcaaggttag gaacagagag
     3061 acagcagaat atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg
     3121 ccaagaacag ttggaacagc agaatatggg ccaaacagga tatctgtggt aagcagttcc
     3181 tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc tcagcagttt
     3241 ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta
     3301 tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct
     3361 caataaaaga gcccacaacc cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc
     3421 cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt
     3481 tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcacatgca
     3541 gcatgtatca aaattaattt ggtttttttt cttaagtatt tacattaaat ggccatagtt
     3601 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3661 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3721 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3781 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3841 taggctccgc acccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3901 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3961 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4021 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4081 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4141 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4201 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4261 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4321 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4381 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4441 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4501 attatcaaaa aggatcttca cctagatcct tttgcggccg gccgcaaatc aatctaaagt
     4561 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4621 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4681 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4741 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4801 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4861 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4921 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4981 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5041 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5101 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5161 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5221 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5281 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5341 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5401 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5461 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5521 atttagaaaa ataaacaaat aggggttccg cgcacatttc
//
