LOCUS       sequence-394263-        4199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for T2A- mCherry- 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 4199)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4199)
  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            "Unpublished"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-mCherry-P2A-NTRv2-pA ORF-1
FEATURES             Location/Qualifiers
     source          1..4199
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..26
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     18..35
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     19..35
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             62..115
                     /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             116..820
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(258..276)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     526..545
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     720..743
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             830..886
                     /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    1576..1710
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1600..1619)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1654..1673
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2147..2938
                     /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      3031..3619
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3520..3539
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3773..3790
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3949..3976)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4068..4154)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc ggtctctagt
       61 cgagggcaga ggcagtctgc tgacatgcgg tgacgtggaa gagaatcccg gccctgtgag
      121 caagggcgag gaggacaaca tggccatcat caaggagttc atgcgcttca aggtgcacat
      181 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
      241 cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg
      301 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
      361 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
      421 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
      481 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
      541 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
      601 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ccgaggtcaa
      661 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
      721 gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg agcgcgccga
      781 gggccgccac tccaccggcg gcatggacga gctgtacaag ggaagcggag ctactaactt
      841 cagcctgctg aagcaggctg gagacgtgga ggagaacccc gggcctatga ctattgttca
      901 agctgcccaa tcccgctact ccaccaaagc ctttgatgct tcgcgcaaat tgcctgaaga
      961 gaaagtcgcg gcagtgaaag agttaatccg catgagtgcg tccagtgtca actcgcaacc
     1021 ttggcatttt attgtcgcga gcagtgaaga gggaaaagcg cgcatcgcca aagcaacaca
     1081 aggtggtttt gctgctaatg agcgcaagat tttggatgct tctcacgttg tggtgttctg
     1141 cgccaaaacg gcgattgatg aagcgtacct actcgacctt ttggaaagcg aagacaaaga
     1201 tggccgctac gccgatgtcg aagcaaaaaa tggcatgcac gctggtcgtt catttttcgt
     1261 caacatgcac cgctttgact tgaaagacgc gcatcactgg atggaaaagc aagtttacct
     1321 caatgtgggg acgctgctat tgggtgcttc tgcgatggag atcgacgcgg tgccaattga
     1381 aggcttcgat gccaaggtgc ttgatgagga gtttggtctg cgtgagaagg gctttaccag
     1441 cgtggtgatt gtgccgctgg gttaccatag cgaagacgat tttaatgcta agctgccaaa
     1501 atcacgttgg tcagcagaga ctgtttttac cgaaatctaa ctcgagtcta gaactatagt
     1561 gagtcgtatt acgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca
     1621 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     1681 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     1741 caggttcagg gggaggtgtg ggaggttttt taattcctca agagacctct agaccagcca
     1801 ggacagaaat gcctcgactt cgctgctacc caaggttgcc gggtgacgca caccgtggaa
     1861 acggatgaag gcacgaaccc agtggacata agcctgttcg gttcgtaagc tgtaatgcaa
     1921 gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg
     1981 cgcagtggcg gttttcatgg cttgttatga ctgttttttt ggggtacagt ctatgcctcg
     2041 ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa
     2101 cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacattatga gggaagcggt
     2161 gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc gccatctcga
     2221 accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc tgaagccaca
     2281 cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc
     2341 tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga ttctccgcgc
     2401 tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc cagctaagcg
     2461 cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct tcgagccagc
     2521 cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata gcgttgcctt
     2581 ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc tatttgaggc
     2641 gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg atgagcgaaa
     2701 tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa tcgcgccgaa
     2761 ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc ccgtcatact
     2821 tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc gcgcagatca
     2881 gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg gcaaataacc
     2941 ctcgagccac ccatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc
     3001 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3061 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3121 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     3181 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3241 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3301 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3361 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3421 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3481 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3541 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3601 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3661 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3721 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3781 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     3841 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     3901 cgcaattaat acgcgtaccg ctagccagga agagtttgta gaaacgcaaa aaggccatcc
     3961 gtcaggatgg ccttctgctt agtttgatgc ctggcagttt atggcgggcg tcctgcccgc
     4021 caccctccgg gccgttgctt cacaacgttc aaatccgctc ccggcggatt tgtcctactc
     4081 aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttc cgactgagcc
     4141 tttcgtttta tttgatgcct ggcagttccc tactctcgcg ttaacgctag catggatgt
//
