LOCUS       sequence-65831-e        6183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid with EGFP expressed from CMV wild-type 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6183)
  AUTHORS   Ferreira JP, Peacock RW, Lawhorn IE, Wang CL
  TITLE     Modulating ectopic gene expression levels by using retroviral 
            vectors equipped with synthetic promoters.
  JOURNAL   Syst Synth Biol. 2011 Dec;5(3-4):131-8. doi: 
            10.1007/s11693-011-9089-0. Epub 2011 Nov 20.
  PUBMED    23205156
REFERENCE   2  (bases 1 to 6183)
  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"; journalName: "Syst Synth 
            Biol."; date: "2011-12"; volume: "5(3-4):131-8. doi"; pages: " 
            10.1007/s11693-011-9089-0. Epub 2011 Nov 20"
COMMENT     Sequence Label: pCOCK CMVwt
FEATURES             Location/Qualifiers
     source          1..6183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     67..86
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        67..85
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          178..407
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     390..409
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      444..453
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(450..452,453..455,456..1169)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(450..452,453..455,456..1166)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(495..516)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(756..775)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1103..1124
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1189..1206)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1195..1419
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1552..1571)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1702..1749
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             1757..2779
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1757..2779
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    2909..3030
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2946..2965)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3000..3019
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3079..3095)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3079..3095)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3092..3114)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3103..3119
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3127..3133,3134..3151,3152..3157))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3172..3193
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3310..3327)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3481..4069)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3561..3580)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4240..5031,5032..5100))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4863..4882
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5101..5205)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5280..5299)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    5559..5583
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        5644..5947
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5948..6151
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6102..6122
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(join(6173..6183,1..14))
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 ttgtacaaag tggttgatat ccagcacagt ggcggccgct cgagtctaga gggcccgcgg
       61 ttcgaataat acgactcact atagggattt aaatttatta acctgcaggc ttagctccct
      121 ttagtgaggg ttaatgtgct ggaattaatt cgctgtctgc gagggccagc tgttggggtg
      181 agtactccct ctcaaaagcg ggcatgactt ctgcgctaag attgtcagtt tccaaaaacg
      241 aggaggattt gatattcacc tggcccgcgg tgatgccttt gagggtggcc gcgtccatct
      301 ggtcagaaaa gacaatcttt ttgttgtcaa gcttgaggtg tggcaggctt gagatctggc
      361 catacacttg agtgacaatg acatccactt tgcctttctc tccacaggtg tccactccca
      421 ggtccaactg caggtcgagc atcgccacca tggtgagcaa gggcgaggag ctgttcaccg
      481 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
      541 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
      601 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
      661 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
      721 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
      781 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
      841 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
      901 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
      961 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1021 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     1081 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1141 ctctcggcat ggacgagctg tacaagtaag tttaaacccg ctgatcagcc tcgactgtgc
     1201 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     1261 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     1321 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     1381 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     1441 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1501 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1561 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1621 ggtcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1681 gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat
     1741 aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     1801 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     1861 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     1921 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     1981 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca
     2041 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc
     2101 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     2161 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     2221 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     2281 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     2341 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     2401 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     2461 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     2521 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     2581 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     2641 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     2701 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     2761 tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga
     2821 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     2881 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     2941 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     3001 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     3061 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3121 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     3181 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3241 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3301 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3361 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3421 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3481 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3541 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3601 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3661 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3721 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3781 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3841 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3901 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     3961 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4021 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4081 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4141 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4201 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4261 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4321 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4381 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     4441 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     4501 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4561 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     4621 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     4681 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     4741 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     4801 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     4861 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4921 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4981 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5041 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     5101 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5161 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5221 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     5281 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     5341 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     5401 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     5461 tatacgcgtt gacattgatt attgactagg cttttgcaaa aagcttggta ccgagctcgg
     5521 atccactagt ccagtgtggt ggaattctgc agatatcaac aagtttgtac aaaaaagcag
     5581 gcttagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     5641 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     5701 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     5761 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     5821 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     5881 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     5941 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     6001 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     6061 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     6121 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tcacccagct
     6181 ttc
//
