LOCUS       sequence-394477-        3440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mini circle middle entry plasmid for GFP-pA with flanking BsaI cut 
            sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3440)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3440)
  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            "Unpublished"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: pMC-ME_EGFP-pA
FEATURES             Location/Qualifiers
     source          1..3440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(162..179)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(333..921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(413..432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1014..1805)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     polyA_signal    2242..2376
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2279..2298)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2333..2352
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(2406..3119,3120..3122,3123..3125))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSAQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2451..2472)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2800..2819
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3059..3080
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3122..3131
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3158..3175)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3158..3174)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3167..3189)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      3238..3324
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      join(3416..3440,1..3)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttttgcgttt ctacaaactc ttcctggcta gcggtacgcg tattaattgc gttgcgctca
       61 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
      121 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
      181 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
      241 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
      301 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
      361 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
      421 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
      481 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
      541 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
      601 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
      661 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
      721 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
      781 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
      841 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
      901 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
      961 tggaacgacg cgtaactcac gttaagggat tttggtcatg ggtggctcga gggttatttg
     1021 ccgactacct tggtgatctc gcctttcacg tagtggacaa attcttccaa ctgatctgcg
     1081 cgcgaggcca agcgatcttc ttcttgtcca agataagcct gtctagcttc aagtatgacg
     1141 ggctgatact gggccggcag gcgctccatt gcccagtcgg cagcgacatc cttcggcgcg
     1201 attttgccgg ttactgcgct gtaccaaatg cgggacaacg taagcactac atttcgctca
     1261 tcgccagccc agtcgggcgg cgagttccat agcgttaagg tttcatttag cgcctcaaat
     1321 agatcctgtt caggaaccgg atcaaagagt tcctccgccg ctggacctac caaggcaacg
     1381 ctatgttctc ttgcttttgt cagcaagata gccagatcaa tgtcgatcgt ggctggctcg
     1441 aagatacctg caagaatgtc attgcgctgc cattctccaa attgcagttc gcgcttagct
     1501 ggataacgcc acggaatgat gtcgtcgtgc acaacaatgg tgacttctac agcgcggaga
     1561 atctcgctct ctccagggga agccgaagtt tccaaaaggt cgttgatcaa agctcgccgc
     1621 gttgtttcat caagccttac ggtcaccgta accagcaaat caatatcact gtgtggcttc
     1681 aggccgccat ccactgcgga gccgtacaaa tgtacggcca gcaacgtcgg ttcgagatgg
     1741 cgctcgatga cgccaactac ctctgatagt tgagtcgata cttcggcgat caccgcttcc
     1801 ctcataatgt ttaactttgt tttagggcga ctgccctgct gcgtaacatc gttgctgctc
     1861 cataacatca aacatcgacc cacggcgtaa cgcgcttgct gcttggatgc ccgaggcata
     1921 gactgtaccc caaaaaaaca gtcataacaa gccatgaaaa ccgccactgc gccgttacca
     1981 ccgctgcgtt cggtcaaggt tctggaccag ttgcgtgagc gcatacgcta cttgcattac
     2041 agcttacgaa ccgaacaggc ttatgtccac tgggttcgtg ccttcatccg tttccacggt
     2101 gtgcgtcacc cggcaacctt gggtagcagc gaagtcgagg catttctgtc ctggctggtc
     2161 tagaggtctc ttgaggaatt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2221 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     2281 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2341 tccaaactca tcaatgtatc ttatcatgtc tggatctacg taatacgact cactatagtt
     2401 ctagcttact tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc
     2461 agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc
     2521 aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag
     2581 tggtcggcga gctgcgcgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg
     2641 atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc
     2701 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
     2761 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
     2821 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
     2881 tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc
     2941 acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc
     3001 ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg
     3061 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
     3121 accatggtgg cgactagaga ccgggcccga gcttaagact ggccgtcgtt ttacaacgtc
     3181 gtgactggga aaacatccat gctagcgtta acgcgagagt agggaactgc caggcatcaa
     3241 ataaaacgaa aggctcagtc ggaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     3301 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgt gaagcaacgg
     3361 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaac taagcagaag
     3421 gccatcctga cggatggcct
//
