LOCUS       sequence-394264-        3492 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for 2a-GFP-pA ORF 2 (with ATG) with
            flanking BsaI cut sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3492)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3492)
  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            "Unpublished"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 with ATG
FEATURES             Location/Qualifiers
     source          1..3492
                     /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             203..259
                     /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(260..262,263..265,266..979)
                     /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(305..326)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(566..585)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     913..934
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1009..1143
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1033..1052)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1087..1106
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1580..2371
                     /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      2464..3052
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2953..2972
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3206..3223
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3382..3409)
                     /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 ccgctacaaa cttctctctg ctgaagcagg ctggagacgt
      241 ggaggagaac cctggaccta tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
      301 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
      361 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
      421 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
      481 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
      541 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
      601 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
      661 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
      721 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
      781 cggcagcgcg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
      841 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
      901 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
      961 ggacgagctg tacaagtaag ctagaactat agtgagtcgt attacgtaga tccagacatg
     1021 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     1081 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     1141 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     1201 ttttaattcc tcaagagacc tctagaccag ccaggacaga aatgcctcga cttcgctgct
     1261 acccaaggtt gccgggtgac gcacaccgtg gaaacggatg aaggcacgaa cccagtggac
     1321 ataagcctgt tcggttcgta agctgtaatg caagtagcgt atgcgctcac gcaactggtc
     1381 cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca tggcttgtta
     1441 tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc
     1501 cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg gcagtcgccc
     1561 taaaacaaag ttaaacatta tgagggaagc ggtgatcgcc gaagtatcga ctcaactatc
     1621 agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg tacatttgta
     1681 cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc tggttacggt
     1741 gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt tggaaacttc
     1801 ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg ttgtgcacga
     1861 cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag aatggcagcg
     1921 caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc tggctatctt
     1981 gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg aggaactctt
     2041 tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa cgctatggaa
     2101 ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg
     2161 gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga
     2221 gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt atcttggaca
     2281 agaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc actacgtgaa
     2341 aggcgagatc accaaggtag tcggcaaata accctcgagc cacccatgac caaaatccct
     2401 taacgtgagt tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2461 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2521 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2581 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     2641 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2701 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2761 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2821 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2881 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2941 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3001 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3061 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3121 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3181 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     3241 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     3301 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatacgcgta ccgctagcca
     3361 ggaagagttt gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttagtttga
     3421 tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg
     3481 ttcaaatccg ct
//
