LOCUS       sequence-73627-e        6998 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 6998)
  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 6998)
  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-ACC/ACC/ACC-mEGFP-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..6998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             61..771
                     /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(100..121)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(361..380)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     708..729
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(807..1381,1382..1384,1385..1393)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(974..991)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1201..1220
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      1402..1411
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1408..2118
                     /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(1553..1571)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1821..1840
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             2174..2767
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(2968..2985)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3139..3727)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3219..3238)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3901..4692,4693..4761))
                     /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     4524..4543
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4762..4866)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5023..5326
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5327..5530
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    5769..6126
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6191..6607
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6537..6559
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6575..6591
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    6617..6991
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     6929..6945
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
ORIGIN
        1 tcgagtaact aactaaacca tgggttgata aaccatgggt tgattaccat gggcgaattc
       61 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      121 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      181 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      241 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
      301 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
      361 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
      421 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
      481 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
      541 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
      601 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
      661 agcacccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
      721 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gttcgaataa
      781 gcatgcatct agggcggcca attccgcccc tctccctccc ccccccctaa cgttactggc
      841 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgtgattttc caccatattg
      901 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
      961 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
     1021 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
     1081 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
     1141 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
     1201 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
     1261 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa
     1321 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa
     1381 gcttgccaca acccgggatc cgccaccatg gtgagcaagg gcgaggagga taacatggcc
     1441 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     1501 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     1561 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     1621 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     1681 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1741 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     1801 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     1861 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     1921 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     1981 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     2041 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     2101 gacgagctgt acaagtaagc ggccgctcga cgataaaata aaagatttta tttagtctcc
     2161 agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc
     2221 cattttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga tcaaggtcag
     2281 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
     2341 ccggctcagg gccaagaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg
     2401 taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtccagcc
     2461 ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac
     2521 cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg
     2581 ctccccgagc tcaataaaag agcccacaac ccctcactcg gggcgccagt cctccgattg
     2641 actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt
     2701 ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc
     2761 tttcatttcc gacttgtggt ctcgctgcct tgggagggtc tcctctgagt gattgactac
     2821 ccgtcagcgg gggtcttcac atgcagcatg tatcaaaatt aatttggttt tttttcttaa
     2881 gtatttacat taaatggcca tagttgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2941 gtttgcgtat tggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3001 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3061 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3121 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3181 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3241 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3301 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3361 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3421 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3481 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3541 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     3601 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3661 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3721 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3781 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3841 ttaaaaatga agtttgcgca aatcaatcta aagtatatat gagtaaactt ggtctgacag
     3901 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     3961 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     4021 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     4081 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     4141 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     4201 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     4261 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     4321 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     4381 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     4441 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     4501 ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct
     4561 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     4621 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     4681 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     4741 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     4801 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     4861 tccgcgcaca tttccccgaa aagtgccacc agctttgctc ttaggagttt cctaatacat
     4921 cccaaactca aatatataaa gcatttgact tgttctatgc cctagttatt aatagtaatc
     4981 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     5041 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     5101 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     5161 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     5221 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     5281 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     5341 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     5401 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     5461 taacaactcc gccccattga cgcaaatggg cggtaggcat gtacggtggg aggtctatat
     5521 aagcagagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc ctccgattga
     5581 ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg
     5641 gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct
     5701 ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc gacccaccac
     5761 cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt gtctatgact
     5821 gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg gcggacccgt
     5881 ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc cagggacttc
     5941 gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg gactctttgg
     6001 tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc taaaacagtt
     6061 cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg cgcgtcttgt
     6121 ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt tgtctgaaaa
     6181 tatcggcccg ggccagactg ttaccactcc cttaagtttg accttaggtc actggaaaga
     6241 tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt gggttacctt
     6301 ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca cctttaaccg
     6361 agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg gacacccaga
     6421 ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc cctgggtcaa
     6481 gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt ctctccccct
     6541 tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca ctccttctct
     6601 aggcgccccc atatggccat atgagatctt atatggggca cccccgcccc ttgtaaactt
     6661 ccctgaccct gacatgacaa gagttactaa cagcccctct ctccaagctc acttacaggc
     6721 tctctactta gtccagcacg aagtctggag acctctggcg gcagcctacc aagaacaact
     6781 ggaccgaccg gtggtacctc acccttaccg agtcggcgac acagtgtggg tccgccgaca
     6841 ccagactaag aacctagaac ctcgctggaa aggaccttac acagtcctgc tgaccacccc
     6901 caccgccctc aaagtagacg gcatcgcagc ttggatacac gccgcccacg tgaaggctgc
     6961 cgaccccggg ggtggaccat cctctagact gccggatc
//
