LOCUS       sequence-243838-        8086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Basic Retroviral backbone with multiple 2a sites, linkers, BFP and 
            and IRES_puromycin from pQCXIP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8086)
  AUTHORS   Singh, Nevil
  TITLE     Multiple 2a Retroviral backbone with Blue Fluorescent Protein and 
            Puromycin resistance
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8086)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pQ2aB
FEATURES             Location/Qualifiers
     source          1..8086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        66..369
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        370..573
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     570..594
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      718..727
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             733..756
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             781..837
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             874..933
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     CDS             943..1638
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     misc_feature    1681..2248
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1842..1859)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2069..2088
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2281..2880
                     /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(2281..2300)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2777..2797
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3254..3679
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(3782..3804)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3940..3960)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3945..4274
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4125..4260
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4296..4315
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4396..4413)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4567..5155)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4647..4666)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5326..6117,6118..6186))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5949..5968
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6187..6291)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6359..6377
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        6477..6856
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6857..7060
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             7061..7236
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    7299..7498
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             join(7699..8086,1..29)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     8045..8067
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     join(8083..8086,1..13)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
ORIGIN
        1 atccagccct cactccttct ctaggcgccg gaattgaaga tctgggggat cgatcctcta
       61 gagtccgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      121 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      181 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      241 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      301 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      361 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      421 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      481 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      541 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      601 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgc
      661 ggccgggaac ggtgcattgg aacggacctg caggaattga tccgcggccg cagatctgcc
      721 accatgggat ccgactacaa agacgatgac gacaagggct ccggaattca acctgcaggt
      781 gccacgaact tctctctgtt aaagcaagca ggagacgtgg aagaaaaccc cggtccctcg
      841 gccggcctag gaggcctcgc tagcggtggc tcccagtgta ccaactatgc cctgctgaag
      901 ctcgccggcg atgtcgagag taaccccggc cctggcgatc gcagcgagct gattaaggag
      961 aacatgcaca tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca
     1021 tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag
     1081 ggcggccctc tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag
     1141 accttcatca accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc
     1201 ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac
     1261 accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca
     1321 tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg
     1381 taccccgctg acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg
     1441 agccatctga tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc
     1501 aagatgcctg gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac
     1561 gagacctacg tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa
     1621 ctggggcaca agcttaattc cggactcaga tctcgagctg ttaacggcga tcgctaaaac
     1681 tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
     1741 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc
     1801 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca
     1861 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac
     1921 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg
     1981 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
     2041 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct
     2101 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg
     2161 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
     2221 ggttttcctt tgaaaaacac gatgataagc ttgccacaac ccacaaggag acgaccttcc
     2281 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta
     2341 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac
     2401 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     2461 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     2521 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     2581 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     2641 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     2701 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     2761 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     2821 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     2881 cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca tggctccgac
     2941 cgaagccacc cggggcggcc ccgccgaccc cgcacccgcc cccgaggccc accgactcta
     3001 gtcgagggct gcagcgctgc agaggccgag tgcagaactg ctccaaaggg acctcaaggc
     3061 tttccgaggg acactaggct gactccatcg agccagtgta gagataagct tatcgattag
     3121 tccaatttgt taaagacagg atatcagtgg tccaggctct agttttgact caacaatatc
     3181 accagctgaa gcctatagag tacgagccat agataaaata aaagatttta tttagtctcc
     3241 agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc
     3301 cattttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga tcaaggtcag
     3361 gaacagatgg aacagggtcg accctagaga accatcagat gtttccaggg tgccccaagg
     3421 acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt
     3481 cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact cggggcgcca
     3541 gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc aataaaccct cttgcagttg
     3601 catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat tgactacccg
     3661 tcagcggggg tctttcattt gggggctcgt ccgggatcgg gagacccctg cccagggacc
     3721 accgacccac caccgggagg taagctggct gcctcgcgcg tttcggtgat gacggtgaaa
     3781 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     3841 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     3901 cccagtcacg tagcgatagc ggagtgtaga tccggctgtg gaatgtgtgt cagttagggt
     3961 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4021 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4081 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4141 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4201 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4261 taggcttttg caaaaagctt actggcttaa ctatgcggca tcagagcaga ttgtactgag
     4321 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     4381 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4441 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4501 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4561 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4621 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4681 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4741 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4801 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4861 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4921 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4981 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     5041 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5101 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5161 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5221 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5281 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5341 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5401 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5461 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5521 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5581 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5641 tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5701 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5761 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5821 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5881 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5941 aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6001 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6061 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6121 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6181 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6241 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6301 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     6361 taggcgtatc acgaggccct ttcgtcttca agaattagct tggccattgc atacgttgta
     6421 tccatatcat aatatgtaca tttatattgg ctcatgtcca acattaccgc catgttgaca
     6481 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     6541 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     6601 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     6661 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     6721 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     6781 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     6841 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     6901 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     6961 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     7021 cggtaggcgt gtacggtggg aggtctatat aagcagagct caataaaaga gcccacaacc
     7081 cctcactcgg cgcgccagtc ttccgataga ctgcgtcgcc cgggtacccg tattcccaat
     7141 aaagcctctt gctgtttgca tccgaatcgt ggtctcgctg ttccttggga gggtctcctc
     7201 tgagtgattg actacccacg acgggggtct ttcatttggg ggctcgtccg ggatttggag
     7261 acccctgccc agggaccacc gacccaccac cgggaggtaa gctggccagc aacttatctg
     7321 tgtctgtccg attgtctagt gtctatgttt gatgttatgc gcctgcgtct gtactagtta
     7381 gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg aacacccggc
     7441 cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc gacctgagga
     7501 agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg agacgagaac
     7561 ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg aagccgcgcg
     7621 tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac tgtgtttctg
     7681 tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac cttaggtcac
     7741 tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa gagacgttgg
     7801 gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg agacggcacc
     7861 tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg cccgcatgga
     7921 cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga cccccctccc
     7981 tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc cgccccgtct
     8041 ctcccccttg aacctcctcg ttcgaccccg cctcgatcct cccttt
//
