LOCUS       sequence-394470-        3493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for 2a-GFP-pA-ORF 3 (with ATG) with
            flanking BsaI cut sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3493)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3493)
  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            "Unpublished"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-3 with ATG
FEATURES             Location/Qualifiers
     source          1..3493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(3..89)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     138..160
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     152..169
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     153..169
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             198..254
                     /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"
     CDS             join(255..257,258..260,261..974)
                     /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(300..321)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(561..580)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     908..929
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1004..1138
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1028..1047)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1082..1101
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1575..2366
                     /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"
     rep_origin      2459..3047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2948..2967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3201..3218
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3377..3404)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag
       61 tcttccgact gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcgttaac
      121 gctagcatgg atgttttccc agtcacgacg ttgtaaaacg acggccagtc ttaagctcgg
      181 gcccggtctc tagtcccgct acaaacttct ctctgctgaa gcaggctgga gacgtggagg
      241 agaaccctgg acctatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      301 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      361 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      421 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      481 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      541 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
      601 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
      661 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
      721 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
      781 gcgcgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
      841 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
      901 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
      961 agctgtacaa gtaagctaga actatagtga gtcgtattac gtagatccag acatgataag
     1021 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     1081 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     1141 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     1201 attcctcaag agacctctag accagccagg acagaaatgc ctcgacttcg ctgctaccca
     1261 aggttgccgg gtgacgcaca ccgtggaaac ggatgaaggc acgaacccag tggacataag
     1321 cctgttcggt tcgtaagctg taatgcaagt agcgtatgcg ctcacgcaac tggtccagaa
     1381 ccttgaccga acgcagcggt ggtaacggcg cagtggcggt tttcatggct tgttatgact
     1441 gtttttttgg ggtacagtct atgcctcggg catccaagca gcaagcgcgt tacgccgtgg
     1501 gtcgatgttt gatgttatgg agcagcaacg atgttacgca gcagggcagt cgccctaaaa
     1561 caaagttaaa cattatgagg gaagcggtga tcgccgaagt atcgactcaa ctatcagagg
     1621 tagttggcgt catcgagcgc catctcgaac cgacgttgct ggccgtacat ttgtacggct
     1681 ccgcagtgga tggcggcctg aagccacaca gtgatattga tttgctggtt acggtgaccg
     1741 taaggcttga tgaaacaacg cggcgagctt tgatcaacga ccttttggaa acttcggctt
     1801 cccctggaga gagcgagatt ctccgcgctg tagaagtcac cattgttgtg cacgacgaca
     1861 tcattccgtg gcgttatcca gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg
     1921 acattcttgc aggtatcttc gagccagcca cgatcgacat tgatctggct atcttgctga
     1981 caaaagcaag agaacatagc gttgccttgg taggtccagc ggcggaggaa ctctttgatc
     2041 cggttcctga acaggatcta tttgaggcgc taaatgaaac cttaacgcta tggaactcgc
     2101 cgcccgactg ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc atttggtaca
     2161 gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca atggagcgcc
     2221 tgccggccca gtatcagccc gtcatacttg aagctagaca ggcttatctt ggacaagaag
     2281 aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt tgtccactac gtgaaaggcg
     2341 agatcaccaa ggtagtcggc aaataaccct cgagccaccc atgaccaaaa tcccttaacg
     2401 tgagttacgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2461 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2521 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2581 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     2641 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2701 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2761 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2821 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2881 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2941 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3001 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3061 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3121 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3181 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     3241 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     3301 ccgactggaa agcgggcagt gagcgcaacg caattaatac gcgtaccgct agccaggaag
     3361 agtttgtaga aacgcaaaaa ggccatccgt caggatggcc ttctgcttag tttgatgcct
     3421 ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca caacgttcaa
     3481 atccgctccc ggc
//
