LOCUS       sequence-476055-        6138 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  To express D2eGFP from a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6138)
  TITLE     Ploski Lab Plasmids 2025
REFERENCE   2  (bases 1 to 6138)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pAAV CMV-d2eGFP-pA
FEATURES             Location/Qualifiers
     source          1..6138
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      281..290
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(287..289,290..292,293..1003,1004..1009,1010..1132)
                     /codon_start=1
                     /product="EGFP destabilized by fusion to residues 422-461 
                     of mouse ornithine decarboxylase, giving an in vivo 
                     half-life of ~2 hours"
                     /label=d2EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKKLSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV
                     "
     primer_bind     complement(332..353)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(593..612)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     940..961
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1303..1322)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1347..1395
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     polyA_signal    1570..2046
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1769..1788)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     polyA_signal    2094..2228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2118..2137)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2172..2191
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   2342..2482
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2342..2471
                     /label=AAV2 ITR
     rep_origin      2557..3012
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2644..2663)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2854..2875
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3029..3048)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3148..3170
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3208..3226)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3294..3398
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3399..3467,3468..4259)
                     /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     complement(3617..3636)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4430..5018
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4919..4938
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5080..5209
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        5285..5588
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5589..5792
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5743..5763
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5789..5813
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          join(5941..6138,1..181)
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
ORIGIN
        1 gcatataaat atttctgcat ataaattgta actgatgtaa gaggtttcat attgctaata
       61 gcagctacaa tccagctacc attctgcttt tattttatgg ttgggataag gctggattat
      121 tctgagtcca agctaggccc ttttgctaat catgttcata cctcttatct tcctcccaca
      181 gctcctgggc aacgtgctgg tctgtgtgct ggcccatcac tttggcaaag aattgggatt
      241 cgaacatcga ttgaattctg cagtcgggga tccaccggtc gccaccatgg tgagcaaggg
      301 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
      361 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
      421 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
      481 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
      541 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
      601 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
      661 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
      721 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
      781 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
      841 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
      901 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
      961 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagaagctta gccatggctt
     1021 cccgccggag gtggaggagc aggatgatgg cacgctgccc atgtcttgtg cccaggagag
     1081 cgggatggac cgtcaccctg cagcctgtgc ttctgctagg atcaatgtgt aggctgcctt
     1141 ctgcggggct tgccttctgg ccatgccctt cttctctccc ttgcacctgt acctcttggt
     1201 ctttgaataa agcctgagta ggaagtgagg gtctagaact agtgtcgacg caagggttcg
     1261 atccctaccg gttagtaatg agtttaaacg ggggaggcta actgaaacac ggaaggagac
     1321 aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacgggtg
     1381 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
     1441 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
     1501 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
     1561 cagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
     1621 gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac
     1681 taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
     1741 gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
     1801 caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct
     1861 cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg
     1921 tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc
     1981 tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
     2041 gtccttctga ttttgtaggt aaccgggccc aatggcgcgc tagataatac gtagatccag
     2101 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     2161 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     2221 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     2281 aggtttttta attcttgcgg ccgcggcggt accggtaacc acgtgcggac cgagcggccg
     2341 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     2401 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     2461 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     2521 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     2581 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     2641 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2701 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2761 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2821 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2881 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     2941 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3001 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3061 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3121 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3181 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3241 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3301 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3361 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3421 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3481 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3541 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3601 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     3661 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     3721 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     3781 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     3841 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     3901 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     3961 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4021 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4081 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4141 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4201 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4261 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4321 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4381 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4441 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4501 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4561 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4621 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4681 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4741 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4801 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4861 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4921 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4981 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5041 tacggttcct ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc
     5101 gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc
     5161 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcacg
     5221 cgtctagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     5281 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     5341 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     5401 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     5461 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     5521 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     5581 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     5641 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     5701 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     5761 gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga
     5821 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgcg
     5881 gattcgaatc ccggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac
     5941 gtaagtaccg cctatagagt ctataggccc acaaaaaatg ctttcttctt ttaatatact
     6001 tttttgttta tcttatttct aatactttcc ctaatctctt tctttcaggg caataatgat
     6061 acaatgtatc atgcctcttt gcaccattct aaagaataac agtgataatt tctgggttaa
     6121 ggcaatagca atatttct
//
