LOCUS       sequence-394270-        3489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for 2a-eGFP-pA ORF2 (without ATG) 
            with flanking BsaI cut sites .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3489)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3489)
  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            "Unpublished"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_2a-EGFP-pA ORF-2 without ATG
FEATURES             Location/Qualifiers
     source          1..3489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     165..184
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     424..445
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(488..544)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(572..589)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(572..588)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(581..603)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      652..738
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      830..857
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1016..1033)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1187..1775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1267..1286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1868..2659)
                     /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    3096..3230
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3133..3152)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3187..3206
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(3260..3489,1..484,485..487))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSAQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3305..3326)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt
       61 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
      121 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
      181 cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
      241 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg
      301 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg
      361 tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt
      421 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
      481 tgctcacagg tccagggttc tcctccacgt ctccagcctg cttcagcaga gagaagtttg
      541 tagcggacta gagaccgggc ccgagcttaa gactggccgt cgttttacaa cgtcgtgact
      601 gggaaaacat ccatgctagc gttaacgcga gagtagggaa ctgccaggca tcaaataaaa
      661 cgaaaggctc agtcggaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
      721 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgtgaagca acggcccgga
      781 gggtggcggg caggacgccc gccataaact gccaggcatc aaactaagca gaaggccatc
      841 ctgacggatg gcctttttgc gtttctacaa actcttcctg gctagcggta cgcgtattaa
      901 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
      961 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     1021 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     1081 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     1141 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     1201 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     1261 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     1321 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     1381 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     1441 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     1501 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     1561 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     1621 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     1681 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     1741 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     1801 ggtctgacgc tcagtggaac gacgcgtaac tcacgttaag ggattttggt catgggtggc
     1861 tcgagggtta tttgccgact accttggtga tctcgccttt cacgtagtgg acaaattctt
     1921 ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg tccaagataa gcctgtctag
     1981 cttcaagtat gacgggctga tactgggccg gcaggcgctc cattgcccag tcggcagcga
     2041 catccttcgg cgcgattttg ccggttactg cgctgtacca aatgcgggac aacgtaagca
     2101 ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat
     2161 ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc gccgctggac
     2221 ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa gatagccaga tcaatgtcga
     2281 tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg ctgccattct ccaaattgca
     2341 gttcgcgctt agctggataa cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt
     2401 ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa aggtcgttga
     2461 tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc aaatcaatat
     2521 cactgtgtgg cttcaggccg ccatccactg cggagccgta caaatgtacg gccagcaacg
     2581 tcggttcgag atggcgctcg atgacgccaa ctacctctga tagttgagtc gatacttcgg
     2641 cgatcaccgc ttccctcata atgtttaact ttgttttagg gcgactgccc tgctgcgtaa
     2701 catcgttgct gctccataac atcaaacatc gacccacggc gtaacgcgct tgctgcttgg
     2761 atgcccgagg catagactgt accccaaaaa aacagtcata acaagccatg aaaaccgcca
     2821 ctgcgccgtt accaccgctg cgttcggtca aggttctgga ccagttgcgt gagcgcatac
     2881 gctacttgca ttacagctta cgaaccgaac aggcttatgt ccactgggtt cgtgccttca
     2941 tccgtttcca cggtgtgcgt cacccggcaa ccttgggtag cagcgaagtc gaggcatttc
     3001 tgtcctggct ggtctagagg tctcttgagg aattaaaaaa cctcccacac ctccccctga
     3061 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     3121 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3181 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tacgtaatac
     3241 gactcactat agttctagct tacttgtaca gctcgtccat gccgagagtg atcccggcgg
     3301 cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcaggg
     3361 cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg
     3421 tgttctgctg gtagtggtcg gcgagctgcg cgctgccgtc ctcgatgttg tggcggatct
     3481 tgaagttca
//
