LOCUS       sequence-394329-        3491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for 2a-eGFP-pA ORF1 (with ATG) with
            flanking BsaI cut sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3491)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3491)
  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            "Unpublished"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 with ATG
FEATURES             Location/Qualifiers
     source          1..3491
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(7..93)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     142..164
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     156..173
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     157..173
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             200..256
                     /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(257..259,260..262,263..976)
                     /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(302..323)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(563..582)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     910..931
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1006..1140
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1030..1049)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1084..1103
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1577..2368
                     /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      2461..3049
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2950..2969
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3203..3220
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3379..3406)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
       61 ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcgt
      121 taacgctagc atggatgttt tcccagtcac gacgttgtaa aacgacggcc agtcttaagc
      181 tcgggcccgg tctctagtcg ctacaaactt ctctctgctg aagcaggctg gagacgtgga
      241 ggagaaccct ggacctatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      301 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
      361 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
      421 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
      481 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
      541 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
      601 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
      661 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
      721 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
      781 cagcgcgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
      841 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
      901 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
      961 cgagctgtac aagtaagcta gaactatagt gagtcgtatt acgtagatcc agacatgata
     1021 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     1081 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     1141 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
     1201 taattcctca agagacctct agaccagcca ggacagaaat gcctcgactt cgctgctacc
     1261 caaggttgcc gggtgacgca caccgtggaa acggatgaag gcacgaaccc agtggacata
     1321 agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca actggtccag
     1381 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga
     1441 ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt
     1501 gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa
     1561 aacaaagtta aacattatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga
     1621 ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg
     1681 ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg ttacggtgac
     1741 cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc
     1801 ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga
     1861 catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa
     1921 tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct
     1981 gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga
     2041 tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc
     2101 gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta
     2161 cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg
     2221 cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga
     2281 agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg
     2341 cgagatcacc aaggtagtcg gcaaataacc ctcgagccac ccatgaccaa aatcccttaa
     2401 cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2461 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2521 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2581 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2641 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2701 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2761 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2821 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2881 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2941 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3001 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3061 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3121 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3181 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     3241 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     3301 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg ctagccagga
     3361 agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt agtttgatgc
     3421 ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt cacaacgttc
     3481 aaatccgctc c
//
