LOCUS       sequence-394642-        3475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for 2a-BFP-pA ORF 3 (without ATG) 
            with flanking BsaI cut sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3475)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3475)
  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            "Unpublished"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-3 without ATG
FEATURES             Location/Qualifiers
     source          1..3475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     129..151
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     143..160
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     144..160
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             189..245
                     /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             246..944
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    977..1111
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1001..1020)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1055..1074
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1548..2339
                     /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      2432..3020
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2921..2940
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3174..3191
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3350..3377)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(join(3469..3475,1..80))
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtcttccgac
       61 tgagcctttc gttttatttg atgcctggca gttccctact ctcgcgttaa cgctagcatg
      121 gatgttttcc cagtcacgac gttgtaaaac gacggccagt cttaagctcg ggcccggtct
      181 ctagtcccgc tacaaacttc tctctgctga agcaggctgg agacgtggag gagaaccctg
      241 gacctagcga gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtgg
      301 acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga
      361 ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta
      421 ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc cccgacttct
      481 tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg
      541 gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca
      601 agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa acactcggct
      661 gggaggcctt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca
      721 tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa catcaagacc acatatagat
      781 ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac tacagactgg
      841 aaagaatcaa ggaggccaac aacgagacgt acgtcgagca gcacgaggtg gcagtggcca
      901 gatactgcga cctccctagc aaactggggc acaagcttaa ttaataagct agaactatag
      961 tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg gacaaaccac
     1021 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     1081 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     1141 tcaggttcag ggggaggtgt gggaggtttt ttaattcctc aagagacctc tagaccagcc
     1201 aggacagaaa tgcctcgact tcgctgctac ccaaggttgc cgggtgacgc acaccgtgga
     1261 aacggatgaa ggcacgaacc cagtggacat aagcctgttc ggttcgtaag ctgtaatgca
     1321 agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc ggtggtaacg
     1381 gcgcagtggc ggttttcatg gcttgttatg actgtttttt tggggtacag tctatgcctc
     1441 gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta tggagcagca
     1501 acgatgttac gcagcagggc agtcgcccta aaacaaagtt aaacattatg agggaagcgg
     1561 tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag cgccatctcg
     1621 aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc ctgaagccac
     1681 acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca acgcggcgag
     1741 ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag attctccgcg
     1801 ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat ccagctaagc
     1861 gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc ttcgagccag
     1921 ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat agcgttgcct
     1981 tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat ctatttgagg
     2041 cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc gatgagcgaa
     2101 atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa atcgcgccga
     2161 aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag cccgtcatac
     2221 ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg cgcgcagatc
     2281 agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc ggcaaataac
     2341 cctcgagcca cccatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt
     2401 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2461 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2521 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     2581 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2641 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2701 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2761 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2821 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2881 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2941 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3001 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3061 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3121 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3181 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     3241 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     3301 acgcaattaa tacgcgtacc gctagccagg aagagtttgt agaaacgcaa aaaggccatc
     3361 cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc gtcctgcccg
     3421 ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat ttgtc
//
