LOCUS       sequence-322056-        6233 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral vector to express cytosolic mCherry and an HA-tagged 
            puromycin selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6233)
  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 6233)
  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-mCherry-P2A-PuroR-HA
FEATURES             Location/Qualifiers
     source          1..6233
                     /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             1974..2681
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2119..2137)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2387..2406
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2754..3350
                     /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(2754..2773)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3250..3270
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3360..3386
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(4337..4354)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4508..5096)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4588..4607)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5260..6051,6052..6120))
                     /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     5883..5902
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6121..6225)
                     /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 accatggtga
     1981 gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca
     2041 tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     2101 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct
     2161 gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg
     2221 acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     2281 acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt
     2341 tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga
     2401 agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga
     2461 agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca
     2521 agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca
     2581 agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg
     2641 agggccgcca ctccaccggc ggcatggacg agctgtacaa gggcagcggc gtcgacgcca
     2701 caaacttcag cctgctgaga caggctggcg acgtggaaga gaatcctgga cctatgaccg
     2761 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     2821 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     2881 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     2941 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     3001 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     3061 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     3121 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     3181 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     3241 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     3301 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc ggcagcggct
     3361 acccttacga tgtgcctgat tacgcctgac ctgcaggttt ctgaagcttg agtacatcga
     3421 ttgcattggg tcgatcgacc gggccgccag cacagtggtc gatcgagggg gggcccgcga
     3481 ttagtccaat ttgttaaaga caggatatca gtggtccagg ctctagtttt gactcaacaa
     3541 tatcaccagc tgaagcctat agagtacgag ccatagatag aataaaagat tttatttagt
     3601 ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct agcttaagta
     3661 acgccatttt gcaaggcatg gaaaatacat aactgagaat agagaagttc agatcaaggt
     3721 taggaacaga gagacagcag aatatgggcc aaacaggata tctgtggtaa gcagttcctg
     3781 ccccggctca gggccaagaa cagttggaac agcagaatat gggccaaaca ggatatctgt
     3841 ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtcccg
     3901 ccctcagcag tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg
     3961 accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc
     4021 tgctccccga gctcaataaa agagcccaca acccctcact cggcgcgcca gtcctccgat
     4081 agactgcgtc gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt
     4141 gtggtctcgc tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg
     4201 tctttcacat gcagcatgta tcaaaattaa tttggttttt tttcttaagt atttacatta
     4261 aatggccata gttgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4321 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4381 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4441 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4501 tggcgttttt ccataggctc cgcacccctg acgagcatca caaaaatcga cgctcaagtc
     4561 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4621 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4681 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4741 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4801 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4861 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4921 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4981 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5041 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5101 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5161 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttgcgg ccggccgcaa
     5221 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5281 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5341 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5401 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5461 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5521 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     5581 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5641 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5701 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5761 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5821 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5881 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5941 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6001 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6061 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6121 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6181 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttc
//
