LOCUS       Exported                6691 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  This a retroviral expression plasmid with a minimal polylinker 
            followed by IRES and puro resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    6691 Bicistronic_ires_puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6691)
  AUTHORS   Agalioti T, Giannou AD, Krontira AC, Kanellakis NI, Kati D, Vreka M,
            Pepe M, Spella M, Lilis I, Zazara DE, Nikolouli E, Spiropoulou N, 
            Papadakis A, Papadia K, Voulgaridis A, Harokopos V, Stamou P, 
            Meiners S, Eickelberg O, Snyder LA, Antimisiaris SG, Kardamakis D, 
            Psallidas I, Marazioti A, Stathopoulos GT
  TITLE     Mutant KRAS promotes malignant pleural effusion formation.
  JOURNAL   Nat Commun. 2017 May 16;8:15205. doi: 10.1038/ncomms15205.
  PUBMED    28508873
REFERENCE   2  (bases 1 to 6691)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6691
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             47..561
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    723..739
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(747..777)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    792..813
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(930..947)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1101..1689)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1181..1200)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1860..2720)
                     /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     2483..2502
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2721..2825)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2893..2911
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2949..2971)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3071..3090
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3284..3306
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3298..3315
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3299..3315
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3514..3533
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             3733..4249
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    4313..4654
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             4721..5137
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     5067..5089
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     5105..5121
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    5183..5735
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5327..5344)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5554..5573
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5747..6346
                     /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(5747..5766)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6243..6263
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6427..6444)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6433..6657
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 atcgataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac
       61 ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga aaatacataa
      121 ctgagaatag agaagttcag atcaaggtta ggaacagaga gacagcagaa tatgggccaa
      181 acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc
      241 agatgcggtc ccgccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca
      301 aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct
      361 gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggcgcg
      421 ccagtcctcc gatagactgc gtcgcccggg tacccgtgta tccaataaac cctcttgcag
      481 ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac
      541 ccgtcagcgg gggtctttca tgggtaacag tttcttgaag ttggagaaca acattctgag
      601 ggtaggagtc gaatattaag taatcctgac tcaattagcc actgttttga atccacatac
      661 tccaatactc ctgaaatagt tcattatgga cagcgcaaaa agagctgggg agaattgtga
      721 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
      781 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
      841 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
      901 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
      961 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     1021 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     1081 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     1141 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1201 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     1261 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     1321 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     1381 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     1441 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     1501 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     1561 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     1621 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1681 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     1741 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     1801 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     1861 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     1921 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     1981 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     2041 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     2101 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     2161 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     2221 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     2281 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     2341 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     2401 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     2461 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     2521 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     2581 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     2641 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     2701 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     2761 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     2821 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     2881 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     2941 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     3001 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     3061 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     3121 ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc
     3181 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     3241 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     3301 aaaacgacgg ccagtgccac gctctccctt atgcgactcc tgcattagga agcagcccag
     3361 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     3421 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     3481 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     3541 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggcgattagt
     3601 ccaatttgtt aaagacagga tatcagtggt ccaggctcta gttttgactc aacaatatca
     3661 ccagctgaag cctatagagt acgagccata gataaaataa aagattttat ttagtctcca
     3721 gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc
     3781 attttgcaag gcatggaaaa tacataactg agaatagaga agttcagatc aaggttagga
     3841 acagagagac agcagaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg
     3901 gctcagggcc aagaacagat ggtccccaga tgcggtcccg ccctcagcag tttctagaga
     3961 accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac
     4021 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa
     4081 agagcccaca acccctcact cggcgcgcca gtcctccgat agactgcgtc gcccgggtac
     4141 ccgtattccc aataaagcct cttgctgttt gcatccgaat cgtggactcg ctgatccttg
     4201 ggagggtctc ctcagattga ttgactgccc acctcggggg tctttcattt ggaggttcca
     4261 ccgagatttg gagacccctg cccagggacc accgaccccc ccgccgggag gtaagctggc
     4321 cagcggtcgt ttcgtgtctg tctctgtctt tgtgcgtgtt tgtgccggca tctaatgttt
     4381 gcgcctgcgt ctgtactagt tagctaacta gctctgtatc tggcggaccc gtggtggaac
     4441 tgacgagttc tgaacacccg gccgcaaccc tgggagacgt cccagggact ttgggggccg
     4501 tttttgtggc ccgacctgag gaagggagtc gatgtggaat ccgaccccgt caggatatgt
     4561 ggttctggta ggagacgaga acctaaaaca gttcccgcct ccgtctgaat ttttgctttc
     4621 ggtttggaac cgaagccgcg cgtcttgtct gctgcagcgc tgcagcatcg ttctgtgttg
     4681 tctctgtctg actgtgtttc tgtatttgtc tgaaaattag ggccagactg ttaccactcc
     4741 cttaagtttg accttaggtc actggaaaga tgtcgagcgg atcgctcaca accagtcggt
     4801 agatgtcaag aagagacgtt gggttacctt ctgctctgca gaatggccaa cctttaacgt
     4861 cggatggccg cgagacggca cctttaaccg agacctcatc acccaggtta agatcaaggt
     4921 cttttcacct ggcccgcatg gacacccaga ccaggtcccc tacatcgtga cctgggaagc
     4981 cttggctttt gacccccctc cctgggtcaa gccctttgta caccctaagc ctccgcctcc
     5041 tcttcctcca tccgccccgt ctctccccct tgaacctcct cgttcgaccc cgcctcgatc
     5101 ctccctttat ccagccctca ctccttctct aggcgccgga attagatctc tcgaggttaa
     5161 cgaattccgc cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt
     5221 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
     5281 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
     5341 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa
     5401 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
     5461 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
     5521 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
     5581 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
     5641 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg
     5701 gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccatga ccgagtacaa
     5761 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc
     5821 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg
     5881 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     5941 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     6001 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     6061 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     6121 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     6181 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
     6241 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     6301 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc cgccccacga
     6361 cccgcagcgc ccgaccgaaa ggagcgcacg accccatgca tcgatgatct agagctcgct
     6421 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     6481 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     6541 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     6601 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     6661 ctgaggcgga aagaacctgc agcccaagct t
//
