LOCUS       sequence-394640-        3473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for 2a-BFP-pA ORF 1 (without ATG) 
            with flanking BsaI cut sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3473)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3473)
  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            "Unpublished"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-BFP-pA ORF-1 without ATG
FEATURES             Location/Qualifiers
     source          1..3473
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..155
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     147..164
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     148..164
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             191..247
                     /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             248..946
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    979..1113
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1003..1022)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1057..1076
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1550..2341
                     /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      2434..3022
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2923..2942
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3176..3193
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3352..3379)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(join(3471..3473,1..84))
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttc
       61 cgactgagcc tttcgtttta tttgatgcct ggcagttccc tactctcgcg ttaacgctag
      121 catggatgtt ttcccagtca cgacgttgta aaacgacggc cagtcttaag ctcgggcccg
      181 gtctctagtc gctacaaact tctctctgct gaagcaggct ggagacgtgg aggagaaccc
      241 tggacctagc gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt
      301 ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca
      361 gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc
      421 tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt
      481 cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg
      541 gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt
      601 caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg
      661 ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga
      721 catggccctg aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag
      781 atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact
      841 ggaaagaatc aaggaggcca acaacgagac gtacgtcgag cagcacgagg tggcagtggc
      901 cagatactgc gacctcccta gcaaactggg gcacaagctt aattaataag ctagaactat
      961 agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc
     1021 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     1081 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     1141 tttcaggttc agggggaggt gtgggaggtt ttttaattcc tcaagagacc tctagaccag
     1201 ccaggacaga aatgcctcga cttcgctgct acccaaggtt gccgggtgac gcacaccgtg
     1261 gaaacggatg aaggcacgaa cccagtggac ataagcctgt tcggttcgta agctgtaatg
     1321 caagtagcgt atgcgctcac gcaactggtc cagaaccttg accgaacgca gcggtggtaa
     1381 cggcgcagtg gcggttttca tggcttgtta tgactgtttt tttggggtac agtctatgcc
     1441 tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag
     1501 caacgatgtt acgcagcagg gcagtcgccc taaaacaaag ttaaacatta tgagggaagc
     1561 ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg agcgccatct
     1621 cgaaccgacg ttgctggccg tacatttgta cggctccgca gtggatggcg gcctgaagcc
     1681 acacagtgat attgatttgc tggttacggt gaccgtaagg cttgatgaaa caacgcggcg
     1741 agctttgatc aacgaccttt tggaaacttc ggcttcccct ggagagagcg agattctccg
     1801 cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa
     1861 gcgcgaactg caatttggag aatggcagcg caatgacatt cttgcaggta tcttcgagcc
     1921 agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac atagcgttgc
     1981 cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg atctatttga
     2041 ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg gcgatgagcg
     2101 aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc
     2161 gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc agcccgtcat
     2221 acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct cgcgcgcaga
     2281 tcagttggaa gaatttgtcc actacgtgaa aggcgagatc accaaggtag tcggcaaata
     2341 accctcgagc cacccatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc
     2401 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2461 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2521 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2581 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2641 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2701 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2761 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2821 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2881 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2941 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3001 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3061 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3121 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3181 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     3241 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     3301 caacgcaatt aatacgcgta ccgctagcca ggaagagttt gtagaaacgc aaaaaggcca
     3361 tccgtcagga tggccttctg cttagtttga tgcctggcag tttatggcgg gcgtcctgcc
     3421 cgccaccctc cgggccgttg cttcacaacg ttcaaatccg ctcccggcgg att
//
