LOCUS       Exported                6969 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Retroviral expression vector. Contains an altered Kozak sequence 
            and/or upstream open reading frames to modulate expression at the 
            level of translation..
ACCESSION   .
VERSION     .
KEYWORDS    pCru5-/CGA-mEGFP-IRES-mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6969)
  AUTHORS   Ferreira JP, Overton KW, Wang CL
  TITLE     Tuning gene expression with synthetic upstream open reading frames.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Jul 9;110(28):11284-9. doi: 
            10.1073/pnas.1305590110. Epub 2013 Jun 24.
  PUBMED    23798422
REFERENCE   2  (bases 1 to 6969)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6969
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             32..742
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(71..92)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(332..351)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     679..700
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    778..1351
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(945..962)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1172..1191
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      1373..1382
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1379..2089
                     /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(1524..1542)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1792..1811
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             2145..2738
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(2939..2956)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3110..3698)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3190..3209)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3872..4732)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4495..4514
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4733..4837)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        4994..5297
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5298..5501
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    5740..6097
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6162..6578
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6508..6530
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6546..6562
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    6588..6962
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     6900..6916
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
ORIGIN
        1 tcgagtaact aactaacgaa tgggcgaatt cagcaagggc gaggagctgt tcaccggggt
       61 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
      121 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      181 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
      241 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      301 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
      361 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
      421 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
      481 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
      541 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
      601 ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc
      661 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
      721 cggcatggac gagctgtaca agttcgaata agcatgcatc tagggcggcc aattccgccc
      781 ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc
      841 gtttgtctat atgtgatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa
      901 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat
      961 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac
     1021 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg
     1081 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt
     1141 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg
     1201 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac
     1261 atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga
     1321 cgtggttttc ctttgaaaaa cacgatgata agcttgccac aacccgggat ccgccaccat
     1381 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     1441 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     1501 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     1561 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     1621 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     1681 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     1741 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     1801 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     1861 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     1921 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     1981 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     2041 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaag cggccgctcg
     2101 acgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc
     2161 tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa
     2221 ctgagaatag agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca
     2281 aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca
     2341 gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca
     2401 agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg
     2461 tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag
     2521 ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa
     2581 cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca
     2641 ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct
     2701 ctgagtgatt gactacccgt cagcgggggt ctttcatttc cgacttgtgg tctcgctgcc
     2761 ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtcttca catgcagcat
     2821 gtatcaaaat taatttggtt ttttttctta agtatttaca ttaaatggcc atagttgcat
     2881 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttggcgctct tccgcttcct
     2941 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3001 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3061 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3121 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3181 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3241 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3301 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3361 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3421 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3481 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3541 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3601 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3661 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3721 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3781 caaaaaggat cttcacctag atccttttaa attaaaaatg aagtttgcgc aaatcaatct
     3841 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3901 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3961 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4021 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4081 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4141 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4201 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4261 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4321 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4381 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4441 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata
     4501 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4561 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4621 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4681 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4741 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4801 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4861 cagctttgct cttaggagtt tcctaataca tcccaaactc aaatatataa agcatttgac
     4921 ttgttctatg ccctagttat taatagtaat caattacggg gtcattagtt catagcccat
     4981 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     5041 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     5101 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     5161 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     5221 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     5281 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     5341 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     5401 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     5461 gcggtaggca tgtacggtgg gaggtctata taagcagagc tcaataaaag agcccacaac
     5521 ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa
     5581 taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc
     5641 tgagtgattg actacccgtc agcgggggtc tttcatttgg gggctcgtcc gggatcggga
     5701 gacccctgcc cagggaccac cgacccacca ccgggaggta agctggccag caacttatct
     5761 gtgtctgtcc gattgtctag tgtctatgac tgattttatg cgcctgcgtc ggtactagtt
     5821 agctaactag ctctgtatct ggcggacccg tggtggaact gacgagttcg gaacacccgg
     5881 ccgcaaccct gggagacgtc ccagggactt cgggggccgt ttttgtggcc cgacctgagt
     5941 ccaaaaatcc cgatcgtttt ggactctttg gtgcaccccc cttagaggag ggatatgtgg
     6001 ttctggtagg agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg
     6061 tttgggaccg aagccgcgcc gcgcgtcttg tctgctgcag catcgttctg tgttgtctct
     6121 gtctgactgt gtttctgtat ttgtctgaaa atatcggccc gggccagact gttaccactc
     6181 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg
     6241 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg
     6301 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg
     6361 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag
     6421 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc
     6481 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat
     6541 cctcccttta tccagccctc actccttctc taggcgcccc catatggcca tatgagatct
     6601 tatatggggc acccccgccc cttgtaaact tccctgaccc tgacatgaca agagttacta
     6661 acagcccctc tctccaagct cacttacagg ctctctactt agtccagcac gaagtctgga
     6721 gacctctggc ggcagcctac caagaacaac tggaccgacc ggtggtacct cacccttacc
     6781 gagtcggcga cacagtgtgg gtccgccgac accagactaa gaacctagaa cctcgctgga
     6841 aaggacctta cacagtcctg ctgaccaccc ccaccgccct caaagtagac ggcatcgcag
     6901 cttggataca cgccgcccac gtgaaggctg ccgaccccgg gggtggacca tcctctagac
     6961 tgccggatc
//
