LOCUS       sequence-394475-        3488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for 2a-eGFP-pA ORF1 (without ATG) 
            with flanking BsaI cut sites .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3488)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3488)
  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            "Unpublished"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-1 without ATG
FEATURES             Location/Qualifiers
     source          1..3488
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(12..98)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     147..169
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     161..178
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     162..178
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             205..261
                     /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(262..264,265..978)
                     /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(304..325)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(565..584)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     912..933
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1008..1142
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1032..1051)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1086..1105
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1579..2370
                     /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      2463..3051
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2952..2971
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3205..3222
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3381..3408)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga
       61 aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt tccctactct
      121 cgcgttaacg ctagcatgga tgttttccca gtcacgacgt tgtaaaacga cggccagtct
      181 taagctcggg cccggtctct agtcgctaca aacttctctc tgctgaagca ggctggagac
      241 gtggaggaga accctggacc tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      301 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      361 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      421 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
      481 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      541 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
      601 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
      661 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
      721 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
      781 ggcagcgcgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
      841 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
      901 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
      961 gacgagctgt acaagtaagc tagaactata gtgagtcgta ttacgtagat ccagacatga
     1021 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     1081 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
     1141 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt
     1201 tttaattcct caagagacct ctagaccagc caggacagaa atgcctcgac ttcgctgcta
     1261 cccaaggttg ccgggtgacg cacaccgtgg aaacggatga aggcacgaac ccagtggaca
     1321 taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg caactggtcc
     1381 agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat ggcttgttat
     1441 gactgttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc gcgttacgcc
     1501 gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg cagtcgccct
     1561 aaaacaaagt taaacattat gagggaagcg gtgatcgccg aagtatcgac tcaactatca
     1621 gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac
     1681 ggctccgcag tggatggcgg cctgaagcca cacagtgata ttgatttgct ggttacggtg
     1741 accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg
     1801 gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac
     1861 gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc
     1921 aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct ggctatcttg
     1981 ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt
     2041 gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac gctatggaac
     2101 tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg
     2161 tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag
     2221 cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta tcttggacaa
     2281 gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa
     2341 ggcgagatca ccaaggtagt cggcaaataa ccctcgagcc acccatgacc aaaatccctt
     2401 aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2461 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2521 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2581 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     2641 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2701 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2761 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2821 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2881 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2941 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3001 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3061 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3121 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3181 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     3241 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     3301 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac cgctagccag
     3361 gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat
     3421 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt
     3481 tcaaatcc
//
