LOCUS       sequence-394645-        4190 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for T2A- BFP- P2A- ntr version 2- 
            pA (ORF 1) with flanking BsaI cut sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4190)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4190)
  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            "Unpublished"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_T2A-BFP-P2A-NTRv2-pA ORF-1
FEATURES             Location/Qualifiers
     source          1..4190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(5..91)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     140..162
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     154..171
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     155..171
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             198..251
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             252..947
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             957..1013
                     /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"
     polyA_signal    1703..1837
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1727..1746)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1781..1800
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2274..3065
                     /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      3158..3746
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3647..3666
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3900..3917
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4076..4103)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
       61 agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgtta
      121 acgctagcat ggatgttttc ccagtcacga cgttgtaaaa cgacggccag tcttaagctc
      181 gggcccggtc tctagtcgag ggcagaggca gtctgctgac atgcggtgac gtggaagaga
      241 atcccggccc tagcgagctg attaaggaga acatgcacat gaagctgtac atggagggca
      301 ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca
      361 cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc
      421 tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg
      481 acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag
      541 acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca
      601 acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac
      661 tcggctggga ggccttcacc gagacgctgt accccgctga cggcggcctg gaaggcagaa
      721 acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacatc aagaccacat
      781 atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca
      841 gactggaaag aatcaaggag gccaacaacg agacgtacgt cgagcagcac gaggtggcag
      901 tggccagata ctgcgacctc cctagcaaac tggggcacaa gcttaatgga agcggagcta
      961 ctaacttcag cctgctgaag caggctggag acgtggagga gaaccccggg cctatgacta
     1021 ttgttcaagc tgcccaatcc cgctactcca ccaaagcctt tgatgcttcg cgcaaattgc
     1081 ctgaagagaa agtcgcggca gtgaaagagt taatccgcat gagtgcgtcc agtgtcaact
     1141 cgcaaccttg gcattttatt gtcgcgagca gtgaagaggg aaaagcgcgc atcgccaaag
     1201 caacacaagg tggttttgct gctaatgagc gcaagatttt ggatgcttct cacgttgtgg
     1261 tgttctgcgc caaaacggcg attgatgaag cgtacctact cgaccttttg gaaagcgaag
     1321 acaaagatgg ccgctacgcc gatgtcgaag caaaaaatgg catgcacgct ggtcgttcat
     1381 ttttcgtcaa catgcaccgc tttgacttga aagacgcgca tcactggatg gaaaagcaag
     1441 tttacctcaa tgtggggacg ctgctattgg gtgcttctgc gatggagatc gacgcggtgc
     1501 caattgaagg cttcgatgcc aaggtgcttg atgaggagtt tggtctgcgt gagaagggct
     1561 ttaccagcgt ggtgattgtg ccgctgggtt accatagcga agacgatttt aatgctaagc
     1621 tgccaaaatc acgttggtca gcagagactg tttttaccga aatctaactc gagtctagaa
     1681 ctatagtgag tcgtattacg tagatccaga catgataaga tacattgatg agtttggaca
     1741 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     1801 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     1861 tatgtttcag gttcaggggg aggtgtggga ggttttttaa ttcctcaaga gacctctaga
     1921 ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg tgacgcacac
     1981 cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt cgtaagctgt
     2041 aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg
     2101 gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg gtacagtcta
     2161 tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga
     2221 gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac attatgaggg
     2281 aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc atcgagcgcc
     2341 atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat ggcggcctga
     2401 agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc
     2461 ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag agcgagattc
     2521 tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg cgttatccag
     2581 ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca ggtatcttcg
     2641 agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga gaacatagcg
     2701 ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa caggatctat
     2761 ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg gctggcgatg
     2821 agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg
     2881 cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag tatcagcccg
     2941 tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg
     3001 cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag gtagtcggca
     3061 aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg tcgttccact
     3121 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3181 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3241 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3301 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3361 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3421 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3481 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3541 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3601 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3661 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3721 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3781 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3841 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3901 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     3961 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     4021 agcgcaacgc aattaatacg cgtaccgcta gccaggaaga gtttgtagaa acgcaaaaag
     4081 gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc
     4141 tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
//
