LOCUS       sequence-394639-        4201 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for T2A- mCherry- 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 4201)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4201)
  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            "Unpublished"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-3
FEATURES             Location/Qualifiers
     source          1..4201
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..28
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     20..37
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     21..37
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             66..119
                     /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             120..824
                     /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(262..280)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     530..549
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     724..747
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             834..890
                     /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    1580..1714
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1604..1623)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1658..1677
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2151..2942
                     /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      3035..3623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3524..3543
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3777..3794
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3953..3980)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4072..4158)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gttttcccag tcacgacgtt gtaaaacgac ggccagtctt aagctcgggc ccggtctcta
       61 gtcccgaggg cagaggcagt ctgctgacat gcggtgacgt ggaagagaat cccggccctg
      121 tgagcaaggg cgaggaggac aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
      181 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
      241 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg
      301 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
      361 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
      421 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
      481 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
      541 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
      601 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgccgagg
      661 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
      721 tcaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgcg
      781 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagggaagc ggagctacta
      841 acttcagcct gctgaagcag gctggagacg tggaggagaa ccccgggcct atgactattg
      901 ttcaagctgc ccaatcccgc tactccacca aagcctttga tgcttcgcgc aaattgcctg
      961 aagagaaagt cgcggcagtg aaagagttaa tccgcatgag tgcgtccagt gtcaactcgc
     1021 aaccttggca ttttattgtc gcgagcagtg aagagggaaa agcgcgcatc gccaaagcaa
     1081 cacaaggtgg ttttgctgct aatgagcgca agattttgga tgcttctcac gttgtggtgt
     1141 tctgcgccaa aacggcgatt gatgaagcgt acctactcga ccttttggaa agcgaagaca
     1201 aagatggccg ctacgccgat gtcgaagcaa aaaatggcat gcacgctggt cgttcatttt
     1261 tcgtcaacat gcaccgcttt gacttgaaag acgcgcatca ctggatggaa aagcaagttt
     1321 acctcaatgt ggggacgctg ctattgggtg cttctgcgat ggagatcgac gcggtgccaa
     1381 ttgaaggctt cgatgccaag gtgcttgatg aggagtttgg tctgcgtgag aagggcttta
     1441 ccagcgtggt gattgtgccg ctgggttacc atagcgaaga cgattttaat gctaagctgc
     1501 caaaatcacg ttggtcagca gagactgttt ttaccgaaat ctaactcgag tctagaacta
     1561 tagtgagtcg tattacgtag atccagacat gataagatac attgatgagt ttggacaaac
     1621 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     1681 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     1741 gtttcaggtt cagggggagg tgtgggaggt tttttaattc ctcaagagac ctctagacca
     1801 gccaggacag aaatgcctcg acttcgctgc tacccaaggt tgccgggtga cgcacaccgt
     1861 ggaaacggat gaaggcacga acccagtgga cataagcctg ttcggttcgt aagctgtaat
     1921 gcaagtagcg tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta
     1981 acggcgcagt ggcggttttc atggcttgtt atgactgttt ttttggggta cagtctatgc
     2041 ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca
     2101 gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatt atgagggaag
     2161 cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc
     2221 tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc
     2281 cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc
     2341 gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc
     2401 gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta
     2461 agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc
     2521 cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg
     2581 ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg
     2641 aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc
     2701 gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc
     2761 cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca
     2821 tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag
     2881 atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat
     2941 aaccctcgag ccacccatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag
     3001 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3061 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3121 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3181 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3241 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3301 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3361 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3421 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3481 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3541 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3601 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3661 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3721 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3781 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     3841 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     3901 gcaacgcaat taatacgcgt accgctagcc aggaagagtt tgtagaaacg caaaaaggcc
     3961 atccgtcagg atggccttct gcttagtttg atgcctggca gtttatggcg ggcgtcctgc
     4021 ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg gatttgtcct
     4081 actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt cttccgactg
     4141 agcctttcgt tttatttgat gcctggcagt tccctactct cgcgttaacg ctagcatgga
     4201 t
//
