LOCUS       sequence-322050-        6253 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 and an HA-tagged puromycin selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6253)
  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 6253)
  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-P2A-PuroR-HA-Rluc_miR
FEATURES             Location/Qualifiers
     source          1..6253
                     /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"
     CDS             2448..3044
                     /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(2448..2467)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2944..2964
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3054..3080
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     ncRNA           3107..3228
                     /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           3256..3270
                     /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           3327..3439
                     /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(4357..4374)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4528..5116)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4608..4627)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5280..6071,6072..6140))
                     /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     5903..5922
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6141..6245)
                     /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 tcctgggcag cggcgtcgac gccacaaact
     2401 tcagcctgct gagacaggct ggcgacgtgg aagagaatcc tggacctatg accgagtaca
     2461 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
     2521 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
     2581 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     2641 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     2701 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     2761 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     2821 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     2881 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     2941 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     3001 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgccggcagc ggctaccctt
     3061 acgatgtgcc tgattacgcc tgacctgcag gtttctgaag ctttgtttga atgaggcttc
     3121 agtactttac agaatcgttg cctgcacatc ttggaaacac ttgctgggat tacttcttca
     3181 ggttaaccca acagaaggct cgagaaggta tattgctgtt gacagtgagc gcaggaatta
     3241 taatgcttat ctatagtgaa gccacagatg tatagataag cattataatt cctatgccta
     3301 ctgcctcgga cttcaagggg ctagaattca aggggctact ttaggagcaa ttatcttgtt
     3361 tactaaaact gaataccttg ctatctcttt gatacatttt tacaaagctg aattaaaatg
     3421 gtataaatta aatcacttta tcgattgcat tgggtcgacc gggccgccag cacagtggtc
     3481 gatcgagggg gggcccgcga ttagtccaat ttgttaaaga caggatatca gtggtccagg
     3541 ctctagtttt gactcaacaa tatcaccagc tgaagcctat agagtacgag ccatagatag
     3601 aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt
     3661 ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaatacat aactgagaat
     3721 agagaagttc agatcaaggt taggaacaga gagacagcag aatatgggcc aaacaggata
     3781 tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagttggaac agcagaatat
     3841 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat
     3901 ggtccccaga tgcggtcccg ccctcagcag tttctagaga accatcagat gtttccaggg
     3961 tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct
     4021 cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact
     4081 cggcgcgcca gtcctccgat agactgcgtc gcccgggtac ccgtgtatcc aataaaccct
     4141 cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat
     4201 tgactacccg tcagcggggg tctttcacat gcagcatgta tcaaaattaa tttggttttt
     4261 tttcttaagt atttacatta aatggccata gttgcattaa tgaatcggcc aacgcgcggg
     4321 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4381 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4441 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4501 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcacccctg acgagcatca
     4561 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4621 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4681 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4741 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4801 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4861 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4921 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4981 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5041 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5101 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5161 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5221 ccttttgcgg ccggccgcaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5281 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5341 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5401 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5461 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5521 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5581 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5641 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5701 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5761 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5821 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5881 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5941 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6001 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6061 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6121 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6181 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6241 ccgcgcacat ttc
//
