LOCUS       sequence-322300-        6242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral vector to express cytosolic mTurquoise2 and an HA-tagged 
            puromycin selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6242)
  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 6242)
  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-mTurquoise2-P2A-PuroR-HA
FEATURES             Location/Qualifiers
     source          1..6242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        81..185
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(186..254,255..1046)
                     /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     complement(404..423)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1210..1798
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1699..1718
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1952..1969
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(2920..2946)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(2956..3552)
                     /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(3036..3056)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3533..3552
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(join(3625..4335,4336..4338,4339..4341))
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3667..3688)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4016..4035
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4275..4296
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4338..4347
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    4408..4786
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     complement(4454..4470)
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             complement(4796..5212)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(4812..4828)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     LTR             join(5700..6242,1..51)
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
ORIGIN
        1 aatggcgtta cttaagctag cttgccaaac ctacaggtgg ggtctttcat tatgcaggtg
       61 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
      121 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      181 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      241 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      301 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      361 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      421 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      481 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      541 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      601 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      661 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      721 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      781 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      841 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      901 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      961 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     1021 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1081 ttgatttgcg gccggccgca aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1141 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1201 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1261 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1321 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     1381 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1441 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1501 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1561 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1621 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1681 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1741 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gtgcggagcc tatggaaaaa
     1801 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     1861 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     1921 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     1981 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcaactatgg
     2041 ccatttaatg taaatactta agaaaaaaaa ccaaattaat tttgatacat gctgcatgtg
     2101 aaagaccccc gctgacgggt agtcaatcac tcagaggaga ccctcccaag gaacagcgag
     2161 accacaagtc ggatgcaact gcaagagggt ttattggata cacgggtacc cgggcgacgc
     2221 agtctatcgg aggactggcg cgccgagtga ggggttgtgg gctcttttat tgagctcggg
     2281 gagcagaagc gcgcgaacag aagcgagaag cgaactgatt ggttagttca aataaggcac
     2341 agggtcattt caggtccttg gggcaccctg gaaacatctg atggttctct agaaactgct
     2401 gagggcggga ccgcatctgg ggaccatctg ttcttggccc tgagccgggg caggaactgc
     2461 ttaccacaga tatcctgttt ggcccatatt ctgctgttcc aactgttctt ggccctgagc
     2521 cggggcagga actgcttacc acagatatcc tgtttggccc atattctgct gtctctctgt
     2581 tcctaacctt gatctgaact tctctattct cagttatgta ttttccatgc cttgcaaaat
     2641 ggcgttactt aagctagctt gccaaaccta caggtggggt ctttcattcc cccctttttc
     2701 tggagactaa ataaaatctt ttattctatc tatggctcgt actctatagg cttcagctgg
     2761 tgatattgtt gagtcaaaac tagagcctgg accactgata tcctgtcttt aacaaattgg
     2821 actaatcgcg ggccccccct cgatcgacca ctgtgctggc ggcccggtcg atcgacccaa
     2881 tgcaatcgat gtactcaagc ttcagaaacc tgcaggtcag gcgtaatcag gcacatcgta
     2941 agggtagccg ctgccggcac cgggcttgcg ggtcatgcac caggtgcgcg gtccttcggg
     3001 cacctcgacg tcggcggtga cggtgaagcc gagccgctcg tagaagggga ggttgcgggg
     3061 cgcggaggtc tccaggaagg cgggcacccc ggcgcgctcg gccgcctcca ctccggggag
     3121 cacgacggcg ctgcccagac ccttgccctg gtggtcgggc gagacgccga cggtggccag
     3181 gaaccacgcg ggctccttgg gccggtgcgg cgccaggagg ccttccatct gttgctgcgc
     3241 ggccagccgg gaaccgctca actcggccat gcgcgggccg atctcggcga acaccgcccc
     3301 cgcttcgacg ctctccggcg tggtccagac cgccaccgcg gcgccgtcgt ccgcgaccca
     3361 caccttgccg atgtcgagcc cgacgcgcgt gaggaagagt tcttgcagct cggtgacccg
     3421 ctcgatgtgg cggtccggat cgacggtgtg gcgcgtggcg gggtagtcgg cgaacgcggc
     3481 ggcgagggtg cgtacggccc tggggacgtc gtcgcgggtg gcgaggcgca ccgtgggctt
     3541 gtactcggtc ataggtccag gattctcttc cacgtcgcca gcctgtctca gcaggctgaa
     3601 gtttgtggcg tcgacgccgc tgcccttgta cagctcgtcc atgccgagag tgatcccggc
     3661 ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag
     3721 cttggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg
     3781 ggtgttctgc tggtagtggt cggcgagctg cacgccgccg tcctcgatgt tgtggcggat
     3841 cttgaagttg gccttgatgc cgttcttctg cttgtcggcg gtgatataga cgttgtcgct
     3901 aaagtagttg tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc
     3961 cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt
     4021 gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt cgggcatggc
     4081 ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cgggcgaagc actgcacgcc
     4141 ccaggacagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat
     4201 gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa
     4261 cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag
     4321 ctcctcgccc ttgctcacca tggtggcacg cgtttccgtg cggccgcacg accggatcca
     4381 cggagttaat taactagcta gatccggcag tctagaggat ggtccacccc cggggtcggc
     4441 agccttcacg tgggcggcgt gtatccaagc tgcgatgccg tctactttga gggcggtggg
     4501 ggtggtcagc aggactgtgt aaggtccttt ccagcgaggt tctaggttct tagtctggtg
     4561 tcggcggacc cacactgtgt cgccgactcg gtaagggtga ggtaccaccg gccggtcggt
     4621 ccagttgttc ttggtaggct gccgccagag gtctccagac ttcgtgctgg actaagtaga
     4681 gagcctgtaa gtgagcttgg agagaggggc tgttagtaac tcttgtcatg tcagggtcag
     4741 ggaagtttac aaggggcggg ggtgccccat ataagatctc atatggccat atgggggcgc
     4801 ctagagaagg agtgagggct ggataaaggg aggatcgagg cggggtcgaa cgaggaggtt
     4861 caagggggag agacggggcg gatggaggaa gaggaggcgg aggcttaggg tgtacaaagg
     4921 gcttgaccca gggagggggg tcaaaagcca aggcttccca ggtcacgatg taggggacct
     4981 ggtctgggtg tccatgcggg ccaggtgaaa agaccttgat cttaacctgg gtgatgaggt
     5041 ctcggttaaa ggtgccgtct cgcggccatc cgacgttaaa ggttggccat tctgcagagc
     5101 agaaggtaac ccaacgtctc ttcttgacat ctaccgactg gttgtgagcg atccgctcga
     5161 catctttcca gtgacctaag gtcaaactta agggagtggt aacagtctgg ccctaatttt
     5221 cagacaaata cagaaacaca gtcagacaga gacaacacag aacgatgctg cagcagacaa
     5281 gacgcgcggc gcggcttcgg tcccaaaccg aaagcaaaaa ttcagacgga ggcgggaact
     5341 gttttaggtt ctcgtctcct accagaacca catatccctc ctattagggg ggtgcaccaa
     5401 agagtccaaa acgatcggga tttttggact caggtcgggc cacaaaaacg gcccccgaag
     5461 tccctgggac gtctcccagg gttgcggccg ggtgttccga actcgtcagt tccaccacgg
     5521 gtccgccaga tacagagcta gttagctaac tagtacagac gcaggcgcaa acattagatg
     5581 ccggcacaaa cacgcacaaa gacagagaca gacacgaaac gaccgctggc cagcttacct
     5641 cccggcgggg gggtcggtgg tccctgggca ggggtctcca aatctcggtg gaacctccaa
     5701 atgaaagacc cccgaggtgg gcagtcaatc aatctgagga gaccctccca aggatcagcg
     5761 agtccacgat tcggatgcaa acagcaagag gctttattgg gaatacgggt acccgggcga
     5821 ctcagtcaat cggaggactg gcgcgccgag tgaggggttg tgggctcttt tattgagctc
     5881 ggggagcaga agcgcgcgaa cagaagcgag aagcgaactg attggttagt tcaaataagg
     5941 cacagggtca tttcaggtcc ttggggcacc ctggaaacat ctgatggttc tctagaaact
     6001 gctgagggct ggaccgcatc tggggaccat ctgttcttgg ccctgagccg gggcaggaac
     6061 tgcttaccac agatatcctg tttggcccat attcagctgt tccatctgtt cttggccctg
     6121 agccggggca ggaactgctt accacagata tcctgtttgg cccatattca gctgttccat
     6181 ctgttcctga ccttgatctg aacttttcta ttctcagtta tgtatttttc catgccttgc
     6241 aa
//
