LOCUS       sequence-73625-e        7013 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 7013)
  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 7013)
  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/ACC-mEGFP-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..7013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             76..786
                     /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(115..136)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(376..395)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     723..744
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(822..1396,1397..1399,1400..1408)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(989..1006)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1216..1235
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      1417..1426
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1423..2133
                     /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(1568..1586)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1836..1855
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             2189..2782
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(2983..3000)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3154..3742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3234..3253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3916..4707,4708..4776))
                     /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     4539..4558
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4777..4881)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5038..5341
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5342..5545
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    5784..6141
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6206..6622
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6552..6574
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6590..6606
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    6632..7006
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     6944..6960
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
ORIGIN
        1 tcgagtaact aactaaacca tgggttgata aaccatgggt tgataaacca tgggttgatt
       61 accatgggcg aattcagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
      121 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
      181 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
      241 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
      301 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
      361 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
      421 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
      481 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
      541 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
      601 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
      661 gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat
      721 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
      781 tacaagttcg aataagcatg catctagggc ggccaattcc gcccctctcc ctcccccccc
      841 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtga
      901 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
      961 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     1021 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     1081 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     1141 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     1201 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     1261 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     1321 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     1381 aaaacacgat gataagcttg ccacaacccg ggatccgcca ccatggtgag caagggcgag
     1441 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     1501 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     1561 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     1621 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     1681 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     1741 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     1801 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     1861 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     1921 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     1981 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     2041 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     2101 tccaccggcg gcatggacga gctgtacaag taagcggccg ctcgacgata aaataaaaga
     2161 ttttatttag tctccagaaa aaggggggaa tgaaagaccc cacctgtagg tttggcaagc
     2221 tagcttaagt aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagagaagt
     2281 tcagatcaag gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg
     2341 taagcagttc ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa
     2401 acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc
     2461 agatgcggtc cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca
     2521 aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct
     2581 gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg
     2641 ccagtcctcc gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag
     2701 ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac
     2761 ccgtcagcgg gggtctttca tttccgactt gtggtctcgc tgccttggga gggtctcctc
     2821 tgagtgattg actacccgtc agcgggggtc ttcacatgca gcatgtatca aaattaattt
     2881 ggtttttttt cttaagtatt tacattaaat ggccatagtt gcattaatga atcggccaac
     2941 gcgcggggag aggcggtttg cgtattggcg ctcttccgct tcctcgctca ctgactcgct
     3001 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3061 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3121 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3181 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3241 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3301 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3361 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3421 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3481 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3541 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     3601 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3661 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3721 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3781 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3841 ctagatcctt ttaaattaaa aatgaagttt gcgcaaatca atctaaagta tatatgagta
     3901 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3961 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4021 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4081 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4141 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4201 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4261 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4321 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4381 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4441 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4501 gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag
     4561 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4621 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4681 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4741 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4801 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4861 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccaccagctt tgctcttagg
     4921 agtttcctaa tacatcccaa actcaaatat ataaagcatt tgacttgttc tatgccctag
     4981 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     5041 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     5101 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     5161 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     5221 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     5281 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     5341 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     5401 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     5461 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcatgtacg
     5521 gtgggaggtc tatataagca gagctcaata aaagagccca caacccctca ctcggggcgc
     5581 cagtcctccg attgactgag tcgcccgggt acccgtgtat ccaataaacc ctcttgcagt
     5641 tgcatccgac ttgtggtctc gctgttcctt gggagggtct cctctgagtg attgactacc
     5701 cgtcagcggg ggtctttcat ttgggggctc gtccgggatc gggagacccc tgcccaggga
     5761 ccaccgaccc accaccggga ggtaagctgg ccagcaactt atctgtgtct gtccgattgt
     5821 ctagtgtcta tgactgattt tatgcgcctg cgtcggtact agttagctaa ctagctctgt
     5881 atctggcgga cccgtggtgg aactgacgag ttcggaacac ccggccgcaa ccctgggaga
     5941 cgtcccaggg acttcggggg ccgtttttgt ggcccgacct gagtccaaaa atcccgatcg
     6001 ttttggactc tttggtgcac cccccttaga ggagggatat gtggttctgg taggagacga
     6061 gaacctaaaa cagttcccgc ctccgtctga atttttgctt tcggtttggg accgaagccg
     6121 cgccgcgcgt cttgtctgct gcagcatcgt tctgtgttgt ctctgtctga ctgtgtttct
     6181 gtatttgtct gaaaatatcg gcccgggcca gactgttacc actcccttaa gtttgacctt
     6241 aggtcactgg aaagatgtcg agcggatcgc tcacaaccag tcggtagatg tcaagaagag
     6301 acgttgggtt accttctgct ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga
     6361 cggcaccttt aaccgagacc tcatcaccca ggttaagatc aaggtctttt cacctggccc
     6421 gcatggacac ccagaccagg tcccctacat cgtgacctgg gaagccttgg cttttgaccc
     6481 ccctccctgg gtcaagccct ttgtacaccc taagcctccg cctcctcttc ctccatccgc
     6541 cccgtctctc ccccttgaac ctcctcgttc gaccccgcct cgatcctccc tttatccagc
     6601 cctcactcct tctctaggcg cccccatatg gccatatgag atcttatatg gggcaccccc
     6661 gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc cctctctcca
     6721 agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc tggcggcagc
     6781 ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg gcgacacagt
     6841 gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac cttacacagt
     6901 cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga tacacgccgc
     6961 ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgccgg atc
//
