LOCUS       sequence-73641-e        6972 bp    DNA     circular SYN 15-DEC-2025
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    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6972)
  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 6972)
  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"; doi: 
            "10.1073/pnas.1305590110"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2013-07-9- 9"; volume: "110"; issue: "28"; pages: "11284-9"
COMMENT     Sequence Label: pCru5-/GCCACC-mEGFP-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..6972
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      17..26
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             35..745
                     /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(74..95)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(335..354)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     682..703
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(781..1355,1356..1358,1359..1367)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(948..965)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1175..1194
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      1376..1385
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1382..2092
                     /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(1527..1545)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1795..1814
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             2148..2741
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(2942..2959)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3113..3701)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3193..3212)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3875..4666,4667..4735))
                     /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     4498..4517
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4736..4840)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        4997..5300
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5301..5504
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    5743..6100
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6165..6581
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6511..6533
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6549..6565
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    6591..6965
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     6903..6919
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
ORIGIN
        1 tcgagtaact aactaagcca ccatgggcga attcagcaag ggcgaggagc tgttcaccgg
       61 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
      121 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
      181 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
      241 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
      301 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
      361 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
      421 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
      481 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
      541 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
      601 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga
      661 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
      721 tctcggcatg gacgagctgt acaagttcga ataagcatgc atctagggcg gccaattccg
      781 cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg
      841 tgcgtttgtc tatatgtgat tttccaccat attgccgtct tttggcaatg tgagggcccg
      901 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
      961 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
     1021 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
     1081 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
     1141 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
     1201 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg
     1261 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg
     1321 ggacgtggtt ttcctttgaa aaacacgatg ataagcttgc cacaacccgg gatccgccac
     1381 catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
     1441 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     1501 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
     1561 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
     1621 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     1681 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     1741 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     1801 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
     1861 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
     1921 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
     1981 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     2041 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt aagcggccgc
     2101 tcgacgataa aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc
     2161 acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaaataca
     2221 taactgagaa tagagaagtt cagatcaagg tcaggaacag atggaacagc tgaatatggg
     2281 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatgga
     2341 acagctgaat atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg
     2401 ccaagaacag atggtcccca gatgcggtcc agccctcagc agtttctaga gaaccatcag
     2461 atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat
     2521 cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca
     2581 caacccctca ctcggggcgc cagtcctccg attgactgag tcgcccgggt acccgtgtat
     2641 ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct
     2701 cctctgagtg attgactacc cgtcagcggg ggtctttcat ttccgacttg tggtctcgct
     2761 gccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct tcacatgcag
     2821 catgtatcaa aattaatttg gttttttttc ttaagtattt acattaaatg gccatagttg
     2881 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattggcgc tcttccgctt
     2941 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3001 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3061 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3121 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3181 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3241 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3301 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3361 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3421 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3481 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3541 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3601 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3661 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3721 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3781 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttg cgcaaatcaa
     3841 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3901 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3961 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4021 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4081 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4141 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4201 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4261 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4321 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4381 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4441 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata
     4501 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4561 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4621 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4681 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4741 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4801 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4861 caccagcttt gctcttagga gtttcctaat acatcccaaa ctcaaatata taaagcattt
     4921 gacttgttct atgccctagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     4981 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     5041 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     5101 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     5161 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     5221 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     5281 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     5341 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     5401 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     5461 tgggcggtag gcatgtacgg tgggaggtct atataagcag agctcaataa aagagcccac
     5521 aacccctcac tcggggcgcc agtcctccga ttgactgagt cgcccgggta cccgtgtatc
     5581 caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc
     5641 ctctgagtga ttgactaccc gtcagcgggg gtctttcatt tgggggctcg tccgggatcg
     5701 ggagacccct gcccagggac caccgaccca ccaccgggag gtaagctggc cagcaactta
     5761 tctgtgtctg tccgattgtc tagtgtctat gactgatttt atgcgcctgc gtcggtacta
     5821 gttagctaac tagctctgta tctggcggac ccgtggtgga actgacgagt tcggaacacc
     5881 cggccgcaac cctgggagac gtcccaggga cttcgggggc cgtttttgtg gcccgacctg
     5941 agtccaaaaa tcccgatcgt tttggactct ttggtgcacc ccccttagag gagggatatg
     6001 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
     6061 cggtttggga ccgaagccgc gccgcgcgtc ttgtctgctg cagcatcgtt ctgtgttgtc
     6121 tctgtctgac tgtgtttctg tatttgtctg aaaatatcgg cccgggccag actgttacca
     6181 ctcccttaag tttgacctta ggtcactgga aagatgtcga gcggatcgct cacaaccagt
     6241 cggtagatgt caagaagaga cgttgggtta ccttctgctc tgcagaatgg ccaaccttta
     6301 acgtcggatg gccgcgagac ggcaccttta accgagacct catcacccag gttaagatca
     6361 aggtcttttc acctggcccg catggacacc cagaccaggt cccctacatc gtgacctggg
     6421 aagccttggc ttttgacccc cctccctggg tcaagccctt tgtacaccct aagcctccgc
     6481 ctcctcttcc tccatccgcc ccgtctctcc cccttgaacc tcctcgttcg accccgcctc
     6541 gatcctccct ttatccagcc ctcactcctt ctctaggcgc ccccatatgg ccatatgaga
     6601 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta
     6661 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct
     6721 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt
     6781 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct
     6841 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg
     6901 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta
     6961 gactgccgga tc
//
