LOCUS       sequence-394647-        4192 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for T2A- BFP- P2A- ntr version 2- 
            pA (ORF 3) with flanking BsaI cut sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4192)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4192)
  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            "Unpublished"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-3
FEATURES             Location/Qualifiers
     source          1..4192
                     /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             200..253
                     /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             254..949
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             959..1015
                     /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    1705..1839
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1729..1748)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1783..1802
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2276..3067
                     /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      3160..3748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3649..3668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3902..3919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4078..4105)
                     /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 tctagtcccg agggcagagg cagtctgctg acatgcggtg acgtggaaga
      241 gaatcccggc cctagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
      301 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
      361 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
      421 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
      481 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
      541 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
      601 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
      661 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag
      721 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac
      781 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
      841 cagactggaa agaatcaagg aggccaacaa cgagacgtac gtcgagcagc acgaggtggc
      901 agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatg gaagcggagc
      961 tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccccg ggcctatgac
     1021 tattgttcaa gctgcccaat cccgctactc caccaaagcc tttgatgctt cgcgcaaatt
     1081 gcctgaagag aaagtcgcgg cagtgaaaga gttaatccgc atgagtgcgt ccagtgtcaa
     1141 ctcgcaacct tggcatttta ttgtcgcgag cagtgaagag ggaaaagcgc gcatcgccaa
     1201 agcaacacaa ggtggttttg ctgctaatga gcgcaagatt ttggatgctt ctcacgttgt
     1261 ggtgttctgc gccaaaacgg cgattgatga agcgtaccta ctcgaccttt tggaaagcga
     1321 agacaaagat ggccgctacg ccgatgtcga agcaaaaaat ggcatgcacg ctggtcgttc
     1381 atttttcgtc aacatgcacc gctttgactt gaaagacgcg catcactgga tggaaaagca
     1441 agtttacctc aatgtgggga cgctgctatt gggtgcttct gcgatggaga tcgacgcggt
     1501 gccaattgaa ggcttcgatg ccaaggtgct tgatgaggag tttggtctgc gtgagaaggg
     1561 ctttaccagc gtggtgattg tgccgctggg ttaccatagc gaagacgatt ttaatgctaa
     1621 gctgccaaaa tcacgttggt cagcagagac tgtttttacc gaaatctaac tcgagtctag
     1681 aactatagtg agtcgtatta cgtagatcca gacatgataa gatacattga tgagtttgga
     1741 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     1801 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     1861 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aattcctcaa gagacctcta
     1921 gaccagccag gacagaaatg cctcgacttc gctgctaccc aaggttgccg ggtgacgcac
     1981 accgtggaaa cggatgaagg cacgaaccca gtggacataa gcctgttcgg ttcgtaagct
     2041 gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga accttgaccg aacgcagcgg
     2101 tggtaacggc gcagtggcgg ttttcatggc ttgttatgac tgtttttttg gggtacagtc
     2161 tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg
     2221 gagcagcaac gatgttacgc agcagggcag tcgccctaaa acaaagttaa acattatgag
     2281 ggaagcggtg atcgccgaag tatcgactca actatcagag gtagttggcg tcatcgagcg
     2341 ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg atggcggcct
     2401 gaagccacac agtgatattg atttgctggt tacggtgacc gtaaggcttg atgaaacaac
     2461 gcggcgagct ttgatcaacg accttttgga aacttcggct tcccctggag agagcgagat
     2521 tctccgcgct gtagaagtca ccattgttgt gcacgacgac atcattccgt ggcgttatcc
     2581 agctaagcgc gaactgcaat ttggagaatg gcagcgcaat gacattcttg caggtatctt
     2641 cgagccagcc acgatcgaca ttgatctggc tatcttgctg acaaaagcaa gagaacatag
     2701 cgttgccttg gtaggtccag cggcggagga actctttgat ccggttcctg aacaggatct
     2761 atttgaggcg ctaaatgaaa ccttaacgct atggaactcg ccgcccgact gggctggcga
     2821 tgagcgaaat gtagtgctta cgttgtcccg catttggtac agcgcagtaa ccggcaaaat
     2881 cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc ctgccggccc agtatcagcc
     2941 cgtcatactt gaagctagac aggcttatct tggacaagaa gaagatcgct tggcctcgcg
     3001 cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg
     3061 caaataaccc tcgagccacc catgaccaaa atcccttaac gtgagttacg cgtcgttcca
     3121 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3181 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3241 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3301 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3361 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3421 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3481 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3541 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3601 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3661 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3721 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3781 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3841 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3901 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     3961 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     4021 tgagcgcaac gcaattaata cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa
     4081 aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt
     4141 cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc cg
//
