LOCUS       sequence-424393-        6242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of EGFP in mouse astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6242)
  TITLE     Brenner lab unpublished plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6242)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pGfa2-EGFP
FEATURES             Location/Qualifiers
     source          1..6242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1822..1831
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1828..1830,1831..1833,1834..2547)
                     /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(1828..1830,1831..1833,1834..2544)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1873..1894)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2134..2153)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2481..2502
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3190..3207)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3190..3206)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3199..3221)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3415..3434)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3534..3556
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3594..3612)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3680..3784
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3785..3853,3854..4645)
                     /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"
     rep_origin      4816..5404
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5305..5324
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5558..5575
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5692..5713
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5728..5733,5734..5751,5752..5758)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5766..5782
                     /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)."
     primer_bind     5771..5793
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5790..5806
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5790..5806
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 agaacatatc ctggtgtgga gtaggggacg ctgctctgac agaggctcgg gggcctgagc
       61 tggctctgtg agctggggag gaggcagaca gccaggcctt gtctgcaagc agacctggca
      121 gcattgggct ggccgccccc cagggcctcc tcttcatgcc cagtgaatga ctcaccttgg
      181 cacagacaca atgttcgggg tgggcacagt gcctgcttcc cgccgcaccc cagcccccct
      241 caaatgcctt ccgagaagcc cattgagcag ggggcttgca ttgcacccca gcctgacagc
      301 ctggcatctt gggataaaag cagcacagcc ccctaggggc tgcccttgct gtgtggcgcc
      361 accggcggtg gagaacaagg ctctattcag cctgtgccca ggaaagggga tcaggggatg
      421 cccaggcatg gacagtgggt ggcagggggg gagaggaggg ctgtctgctt cccagaagtc
      481 caaggacaca aatgggtgag gggactgggc agggttctga ccctgtggga ccagagtgga
      541 gggcgtagat ggacctgaag tctccaggga caacagggcc caggtctcag gctcctagtt
      601 gggcccagtg gctccagcgt ttccaaaccc atccatcccc agaggttctt cccatctctc
      661 caggctgatg tgtgggaact cgaggaaata aatctccagt gggagacgga ggggtggcca
      721 gggaaacggg gcgctgcagg aataaagacg agccagcaca gccagctcat gtgtaacggc
      781 tttgtggagc tgtcaaggcc tggtctctgg gagagaggca cagggaggcc agacaaggaa
      841 ggggtgacct ggagggacag atccaggggc taaagtcctg ataaggcaag agagtgccgg
      901 ccccctcttg ccctatcagg acctccactg ccacatagag gccatgattg acccttagac
      961 aaagggctgg tgtccaatcc cagcccccag ccccagaact ccagggaatg aatgggcaga
     1021 gagcaggaat gtgggacatc tgtgttcaag ggaaggactc caggagtctg ctgggaatga
     1081 ggcctagtag gaaatgaggt ggcccttgag ggtacagaac aggttcattc ttcgccaaat
     1141 tcccagcacc ttgcaggcac ttacagctga gtgagataat gcctgggtta tgaaatcaaa
     1201 aagttggaaa gcaggtcaga ggtcatctgg tacagccctt ccttcccttt tttttttttt
     1261 tttttgtgag acaaggtctc tctctgttgc ccaggctgga gtggcgcaaa cacagctcac
     1321 tgcagcctca acctactggg ctcaagcaat cctccagcct cagcctccca aagtgctggg
     1381 attacaagca tgagccaccc cactcagccc tttccttcct ttttaattga tgcataataa
     1441 ttgtaagtat tcatcatggt ccaaccaacc ctttcttgac ccaccttcct agagagaggg
     1501 tcctcttgct tcagcggtca gggccccaga cccatggtct ggctccaggt accacctgcc
     1561 tcatgcagga gttggcgtgc ccaggaagct ctgcctctgg gcacagtgac ctcagtgggg
     1621 tgaggggagc tctccccata gctgggctgc ggcccaaccc caccccctca ggctatgcca
     1681 gggggtgttg ccaggggcac ccgggcatcg ccagtctagc ccactccttc ataaagccct
     1741 cgcatcccag gagcgagcag agccagagca ggttggagag gagacgcatc acctccgctg
     1801 ctcgcgggga gggtaccggt cgccaccatg gtgagcaagg gcgaggagct gttcaccggg
     1861 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1921 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1981 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     2041 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2101 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2161 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2221 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     2281 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     2341 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     2401 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     2461 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     2521 ctcggcatgg acgagctgta caagtaaagc ggccgcgact ctagaattcc aactgagcgc
     2581 cggtcgctac cattaccaac ttgtctggtg tcaaaaataa taggtccgca gatcccggcc
     2641 agataccgat gctgccgcag caaaagcagg agcagatgcc gccgtcgcag gcgaagatgt
     2701 cgcagacgga ggaggcgatg ctgccggcgg aggaggcgaa gtaagtagag ggctgggctg
     2761 ggctgtgggg ggtgtggggt gcgggactgg gcagtctggg agtccctctc accacttttc
     2821 ttacctttct aggatgctgc cgtcgccgcc gctcatacac cataaggtgt aaaaaatact
     2881 agatgcacag aatagcaagt ccatcaaaac tcctgcgtga gaattttacc agacttcaag
     2941 agcatctcgc cacatcttga aaaatgccac cgtccgatga aaaacaggag cctgctaagg
     3001 aacaatgcca cctgtcaata aatgttgaaa actcatccca ttcctgcctc ttggtccttg
     3061 ggcttgggga ggggtgcgcg gatgtggtta gggaacatga ctggtcaaat gggaagggct
     3121 tcaaaagaat tcccaatatt gactaccaag ccacctgtac agatctgcct gcaggcatgc
     3181 aagcttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     3241 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     3301 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     3361 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     3421 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     3481 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3541 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     3601 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     3661 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3721 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3781 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3841 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3901 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     3961 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4021 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     4081 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     4141 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4201 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4261 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4321 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4381 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4441 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4501 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4561 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4621 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     4681 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4741 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4801 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     4861 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     4921 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     4981 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5041 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5101 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5161 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5221 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5281 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5341 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5401 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5461 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     5521 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     5581 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     5641 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     5701 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     5761 tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga ttacgaattc
     5821 gagctcggta ccagatctga gctcccacct ccctctctgt gctgggactc acagagggag
     5881 acctcaggag gcagtctgtc catcacatgt ccaaatgcag agcataccct gggctgggcg
     5941 cagtggcgca caactgtaat tccagcactt tgggaggctg atgtggaagg atcacttgag
     6001 cccagaagtt ctagaccagc ctgggcaaca tggcaagacc ctatctctac aaaaaaagtt
     6061 aaaaaatcag ccacgtgtgg tgacacacac ctgtagtccc agctattcag gaggctgagg
     6121 tgaggggatc acttaaggct gggaggttga ggctgcagtg agtcgtggtt gcgccactgc
     6181 actccagcct gggcaacagt gagaccctgt ctcaaaagac aaaaaaaaaa aaaaaaaaaa
     6241 aa
//
