LOCUS       Exported                7451 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  This a retroviral expression plasmid with a minimal polylinker 
            followed by IRES and hygro resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    7749 Bicistronic_ires_hygro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7451)
  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 7451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7451
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    34..586
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(178..195)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     405..424
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             622..1659
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
                     MGEAN"
     primer_bind     complement(1657..1676)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1701..1749
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(2188..2207)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2189..2207)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2217..2233)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             2355..2869
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3031..3047
                     /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(3055..3085)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3100..3121
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3238..3255)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3409..3997)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3489..3508)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4168..5028)
                     /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     4791..4810
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5029..5133)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5201..5219
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5257..5279)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5379..5398
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5607..5623
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5822..5841
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             6041..6557
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    6621..6962
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             7029..7445
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     7375..7397
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     7413..7429
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
ORIGIN
        1 agatctctcg aggttaaccg cccccccccc ctaacgttac tggccgaagc cgcttggaat
       61 aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg
      121 tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc
      181 tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt
      241 cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg
      301 acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac
      361 cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg
      421 tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg
      481 ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc
      541 cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaagattc
      601 gaggttgggg ggcaatgaga tatgaaaaag cctgaactca ccgcgacgtc tgtcgagaag
      661 tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa
      721 tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc
      781 gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg
      841 attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc
      901 cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag
      961 ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc
     1021 ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg
     1081 attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc
     1141 gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac
     1201 ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg
     1261 gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc
     1321 ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat
     1381 ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa
     1441 ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc
     1501 gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc
     1561 gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc
     1621 agcactcgtc cggatcggga gatgggggag gctaactgaa acacggaagg agacaatacc
     1681 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg ggtgttgggt
     1741 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     1801 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     1861 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagccactg
     1921 gccccgtggg ttagggacgg ggtcccccat ggggaatggt ttatggttcg tgggggttat
     1981 tattttgggc gttgcgtggg gtcaggtcca cgactggact gagcagacag acccatggtt
     2041 tttggatggc ctgggcatgg accgcatgta ctggcgcgac acgaacaccg ggcgtctgtg
     2101 gctgccaaac acccccgacc cccaaaaacc accgcgcgga tttctggcgt gccaagctag
     2161 tcgagggggg gcccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     2221 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     2281 gcagcacatc cccctttcgc cagagcttat cgataaaata aaagatttta tttagtctcc
     2341 agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc
     2401 cattttgcaa ggcatggaaa atacataact gagaatagag aagttcagat caaggttagg
     2461 aacagagaga cagcagaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
     2521 ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag
     2581 aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa
     2641 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa
     2701 aagagcccac aacccctcac tcggcgcgcc agtcctccga tagactgcgt cgcccgggta
     2761 cccgtgtatc caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg
     2821 ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttcatg ggtaacagtt
     2881 tcttgaagtt ggagaacaac attctgaggg taggagtcga atattaagta atcctgactc
     2941 aattagccac tgttttgaat ccacatactc caatactcct gaaatagttc attatggaca
     3001 gcgcaaaaag agctggggag aattgtgaaa ttgttatccg ctcacaattc cacacaacat
     3061 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3121 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3181 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3241 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3301 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3361 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3421 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3481 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3541 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3601 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3661 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3721 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3781 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3841 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3901 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3961 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4021 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4081 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4141 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4201 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4261 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4321 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4381 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4441 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4501 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4561 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4621 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4681 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4741 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4801 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4861 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4921 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4981 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5041 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5101 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5161 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     5221 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     5281 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     5341 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     5401 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     5461 caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     5521 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     5581 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgccacgc tctcccttat
     5641 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     5701 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     5761 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     5821 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     5881 cgatgcgtcc ggcgtagagg cgattagtcc aatttgttaa agacaggata tcagtggtcc
     5941 aggctctagt tttgactcaa caatatcacc agctgaagcc tatagagtac gagccataga
     6001 taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta
     6061 ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaata cataactgag
     6121 aatagagaag ttcagatcaa ggttaggaac agagagacag cagaatatgg gccaaacagg
     6181 atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg
     6241 cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac
     6301 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg
     6361 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gcgcgccagt
     6421 cctccgatag actgcgtcgc ccgggtaccc gtattcccaa taaagcctct tgctgtttgc
     6481 atccgaatcg tggactcgct gatccttggg agggtctcct cagattgatt gactgcccac
     6541 ctcgggggtc tttcatttgg aggttccacc gagatttgga gacccctgcc cagggaccac
     6601 cgaccccccc gccgggaggt aagctggcca gcggtcgttt cgtgtctgtc tctgtctttg
     6661 tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct gtactagtta gctaactagc
     6721 tctgtatctg gcggacccgt ggtggaactg acgagttctg aacacccggc cgcaaccctg
     6781 ggagacgtcc cagggacttt gggggccgtt tttgtggccc gacctgagga agggagtcga
     6841 tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg agacgagaac ctaaaacagt
     6901 tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg aagccgcgcg tcttgtctgc
     6961 tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac tgtgtttctg tatttgtctg
     7021 aaaattaggg ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg
     7081 tcgagcggat cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct
     7141 gctctgcaga atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag
     7201 acctcatcac ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc
     7261 aggtccccta catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc
     7321 cctttgtaca ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg
     7381 aacctcctcg ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag
     7441 gcgccggaat t
//
