LOCUS       sequence-322055-        6161 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 a DYK-tagged Blasticidin selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6161)
  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 6161)
  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-CD90.2-P2A-BlastDYK-Rluc_miR
FEATURES             Location/Qualifiers
     source          1..6161
                     /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"
     CDS             2961..2984
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     ncRNA           3011..3132
                     /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           3160..3174
                     /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           3231..3343
                     /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(4265..4282)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4436..5024)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4516..4535)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5188..5979,5980..6048))
                     /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     5811..5830
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6049..6153)
                     /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 accatgaacc
     1981 ccgccatctc tgtggccctg ctgctgtctg tgctgcaggt gtccagaggc cagaaagtga
     2041 ccagcctgac cgcctgcctc gtgaaccaga acctgagact ggactgcaga cacgagaaca
     2101 acaccaagga caacagcatc cagcacgagt tctccctgac ccgcgagaag agaaaacacg
     2161 tgctgagcgg caccctgggc atccccgagc acacctacag atccagagtg accctgagca
     2221 accagcccta catcaaggtg ctgaccctgg ccaacttcac cacaaaggac gagggcgact
     2281 acttctgcga actgcaggtg tcaggcgcca accccatgag cagcaacaag agcatcagcg
     2341 tgtacagaga caagctcgtg aagtgcggcg gcatcagcct gctggtgcag aacacctctt
     2401 ggatgctgtt gctgctgctg agcctgagcc tgctgcaggc cctggacttc atctctctgg
     2461 gcagcggcgt cgacgccaca aacttcagcc tgctgagaca ggctggcgac gtggaagaga
     2521 atcctggacc tatgaaaaca tttaacattt ctcaacaaga tctagaatta gtagaagtag
     2581 cgacagagaa gattacaatg ctttatgagg ataataaaca tcatgtggga gcggcaattc
     2641 gtacgaaaac aggagaaatc atttcggcag tacatattga agcgtatata ggacgagtaa
     2701 ctgtttgtgc agaagccatt gcgattggta gtgcagtttc gaatggacaa aaggattttg
     2761 acacgattgt agctgttaga cacccttatt ctgacgaagt agatagaagt attcgagtgg
     2821 taagtccttg tggtatgtgt agggagttga tttcagacta tgcaccagat tgttttgtgt
     2881 taatagaaat gaatggcaag ttagtcaaaa ctacgattga agaactcatt ccactcaaat
     2941 atacccgaaa tggcagcggc gactacaagg acgacgacga caaatgacct gcaggtttct
     3001 gaagctttgt ttgaatgagg cttcagtact ttacagaatc gttgcctgca catcttggaa
     3061 acacttgctg ggattacttc ttcaggttaa cccaacagaa ggctcgagaa ggtatattgc
     3121 tgttgacagt gagcgcagga attataatgc ttatctatag tgaagccaca gatgtataga
     3181 taagcattat aattcctatg cctactgcct cggacttcaa ggggctagaa ttcaaggggc
     3241 tactttagga gcaattatct tgtttactaa aactgaatac cttgctatct ctttgataca
     3301 tttttacaaa gctgaattaa aatggtataa attaaatcac tttatcgatt gcattgggtc
     3361 gatcgaccgg gccgccagca cagtggtcga tcgagggggg gcccgcgatt agtccaattt
     3421 gttaaagaca ggatatcagt ggtccaggct ctagttttga ctcaacaata tcaccagctg
     3481 aagcctatag agtacgagcc atagatagaa taaaagattt tatttagtct ccagaaaaag
     3541 gggggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc
     3601 aaggcatgga aaatacataa ctgagaatag agaagttcag atcaaggtta ggaacagaga
     3661 gacagcagaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg
     3721 gccaagaaca gttggaacag cagaatatgg gccaaacagg atatctgtgg taagcagttc
     3781 ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtcccgcc ctcagcagtt
     3841 tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt
     3901 atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc
     3961 tcaataaaag agcccacaac ccctcactcg gcgcgccagt cctccgatag actgcgtcgc
     4021 ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg
     4081 ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcacatgc
     4141 agcatgtatc aaaattaatt tggttttttt tcttaagtat ttacattaaa tggccatagt
     4201 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4261 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4321 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4381 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4441 ataggctccg cacccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4501 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4561 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4621 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4681 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4741 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4801 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4861 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4921 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4981 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5041 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5101 gattatcaaa aaggatcttc acctagatcc ttttgcggcc ggccgcaaat caatctaaag
     5161 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5221 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5281 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5341 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5401 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5461 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5521 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5581 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5641 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5701 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5761 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5821 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5881 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5941 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6001 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6061 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6121 tatttagaaa aataaacaaa taggggttcc gcgcacattt c
//
