LOCUS       Exported                6983 bp ds-DNA     circular SYN 13-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-CCC/ACC-mEGFP-IRES-mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6983)
  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 6983)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6983
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             46..756
                     /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(85..106)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(346..365)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     693..714
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    792..1365
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(959..976)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1186..1205
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      1387..1396
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1393..2103
                     /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(1538..1556)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1806..1825
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             2159..2752
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(2953..2970)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3124..3712)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3204..3223)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3886..4746)
                     /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     4509..4528
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4747..4851)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5008..5311
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5312..5515
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    5754..6111
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6176..6592
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6522..6544
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6560..6576
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    6602..6976
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     6914..6930
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
ORIGIN
        1 tcgagtaact aactaaccca tgggttgatt accatgggcg aattcagcaa gggcgaggag
       61 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      121 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
      181 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
      241 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
      301 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
      361 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
      421 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
      481 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
      541 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
      601 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccaag
      661 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
      721 gccgggatca ctctcggcat ggacgagctg tacaagttcg aataagcatg catctagggc
      781 ggccaattcc gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa
      841 taaggccggt gtgcgtttgt ctatatgtga ttttccacca tattgccgtc ttttggcaat
      901 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct
      961 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct
     1021 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc
     1081 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa
     1141 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc
     1201 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg
     1261 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc
     1321 ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataagcttg ccacaacccg
     1381 ggatccgcca ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc
     1441 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
     1501 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
     1561 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
     1621 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
     1681 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
     1741 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     1801 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     1861 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     1921 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     1981 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     2041 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     2101 taagcggccg ctcgacgata aaataaaaga ttttatttag tctccagaaa aaggggggaa
     2161 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
     2221 ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag
     2281 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
     2341 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
     2401 ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag
     2461 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
     2521 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
     2581 aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg
     2641 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
     2701 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttccgactt
     2761 gtggtctcgc tgccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc
     2821 ttcacatgca gcatgtatca aaattaattt ggtttttttt cttaagtatt tacattaaat
     2881 ggccatagtt gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattggcg
     2941 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3001 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3061 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3121 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3181 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3241 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3301 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3361 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3421 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3481 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3541 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     3601 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3661 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3721 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3781 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3841 gcgcaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3901 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3961 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4021 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4081 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4141 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4201 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4261 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4321 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4381 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4441 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4501 aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4561 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4621 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4681 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4741 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4801 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4861 ccgaaaagtg ccaccagctt tgctcttagg agtttcctaa tacatcccaa actcaaatat
     4921 ataaagcatt tgacttgttc tatgccctag ttattaatag taatcaatta cggggtcatt
     4981 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     5041 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     5101 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     5161 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     5221 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     5281 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     5341 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     5401 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     5461 attgacgcaa atgggcggta ggcatgtacg gtgggaggtc tatataagca gagctcaata
     5521 aaagagccca caacccctca ctcggggcgc cagtcctccg attgactgag tcgcccgggt
     5581 acccgtgtat ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt
     5641 gggagggtct cctctgagtg attgactacc cgtcagcggg ggtctttcat ttgggggctc
     5701 gtccgggatc gggagacccc tgcccaggga ccaccgaccc accaccggga ggtaagctgg
     5761 ccagcaactt atctgtgtct gtccgattgt ctagtgtcta tgactgattt tatgcgcctg
     5821 cgtcggtact agttagctaa ctagctctgt atctggcgga cccgtggtgg aactgacgag
     5881 ttcggaacac ccggccgcaa ccctgggaga cgtcccaggg acttcggggg ccgtttttgt
     5941 ggcccgacct gagtccaaaa atcccgatcg ttttggactc tttggtgcac cccccttaga
     6001 ggagggatat gtggttctgg taggagacga gaacctaaaa cagttcccgc ctccgtctga
     6061 atttttgctt tcggtttggg accgaagccg cgccgcgcgt cttgtctgct gcagcatcgt
     6121 tctgtgttgt ctctgtctga ctgtgtttct gtatttgtct gaaaatatcg gcccgggcca
     6181 gactgttacc actcccttaa gtttgacctt aggtcactgg aaagatgtcg agcggatcgc
     6241 tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct ctgcagaatg
     6301 gccaaccttt aacgtcggat ggccgcgaga cggcaccttt aaccgagacc tcatcaccca
     6361 ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg tcccctacat
     6421 cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct ttgtacaccc
     6481 taagcctccg cctcctcttc ctccatccgc cccgtctctc ccccttgaac ctcctcgttc
     6541 gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg cccccatatg
     6601 gccatatgag atcttatatg gggcaccccc gccccttgta aacttccctg accctgacat
     6661 gacaagagtt actaacagcc cctctctcca agctcactta caggctctct acttagtcca
     6721 gcacgaagtc tggagacctc tggcggcagc ctaccaagaa caactggacc gaccggtggt
     6781 acctcaccct taccgagtcg gcgacacagt gtgggtccgc cgacaccaga ctaagaacct
     6841 agaacctcgc tggaaaggac cttacacagt cctgctgacc acccccaccg ccctcaaagt
     6901 agacggcatc gcagcttgga tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg
     6961 accatcctct agactgccgg atc
//
