LOCUS       sequence-394330-        3490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for 2a-eGFP-pA ORF3 (without ATG) 
            with flanking BsaI cut sites .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3490)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3490)
  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            "Unpublished"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 without ATG
FEATURES             Location/Qualifiers
     source          1..3490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(9..95)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     144..166
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     158..175
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     159..175
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             204..260
                     /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(261..263,264..977)
                     /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(303..324)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(564..583)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     911..932
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1007..1141
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1031..1050)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1085..1104
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1578..2369
                     /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      2462..3050
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2951..2970
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3204..3221
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3380..3407)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cccggcggat ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag
       61 gcccagtctt ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc
      121 gttaacgcta gcatggatgt tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa
      181 gctcgggccc ggtctctagt cccgctacaa acttctctct gctgaagcag gctggagacg
      241 tggaggagaa ccctggacct gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      301 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
      361 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      421 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
      481 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      541 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
      601 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
      661 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
      721 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
      781 gcagcgcgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
      841 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
      901 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
      961 acgagctgta caagtaagct agaactatag tgagtcgtat tacgtagatc cagacatgat
     1021 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     1081 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     1141 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     1201 ttaattcctc aagagacctc tagaccagcc aggacagaaa tgcctcgact tcgctgctac
     1261 ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa ggcacgaacc cagtggacat
     1321 aagcctgttc ggttcgtaag ctgtaatgca agtagcgtat gcgctcacgc aactggtcca
     1381 gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg
     1441 actgtttttt tggggtacag tctatgcctc gggcatccaa gcagcaagcg cgttacgccg
     1501 tgggtcgatg tttgatgtta tggagcagca acgatgttac gcagcagggc agtcgcccta
     1561 aaacaaagtt aaacattatg agggaagcgg tgatcgccga agtatcgact caactatcag
     1621 aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta catttgtacg
     1681 gctccgcagt ggatggcggc ctgaagccac acagtgatat tgatttgctg gttacggtga
     1741 ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg
     1801 cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt gtgcacgacg
     1861 acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa tggcagcgca
     1921 atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg gctatcttgc
     1981 tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag gaactctttg
     2041 atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg ctatggaact
     2101 cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt
     2161 acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg gcaatggagc
     2221 gcctgccggc ccagtatcag cccgtcatac ttgaagctag acaggcttat cttggacaag
     2281 aagaagatcg cttggcctcg cgcgcagatc agttggaaga atttgtccac tacgtgaaag
     2341 gcgagatcac caaggtagtc ggcaaataac cctcgagcca cccatgacca aaatccctta
     2401 acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2461 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2521 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     2581 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     2641 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     2701 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     2761 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     2821 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     2881 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     2941 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3001 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3061 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3121 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3181 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     3241 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     3301 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tacgcgtacc gctagccagg
     3361 aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg gccttctgct tagtttgatg
     3421 cctggcagtt tatggcgggc gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt
     3481 caaatccgct
//
