LOCUS       sequence-394272-        4200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for T2A- mCherry- P2A- ntr version 
            2- pA (ORF 2) with flanking BsaI cut sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4200)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4200)
  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            "Unpublished"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-2
FEATURES             Location/Qualifiers
     source          1..4200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(76..129)
                     /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"
     primer_bind     complement(157..174)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(157..173)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(166..188)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      237..323
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      415..442
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(601..618)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(772..1360)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(852..871)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1453..2244)
                     /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"
     polyA_signal    2681..2815
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2718..2737)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2772..2791
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(3505..3561)
                     /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             complement(join(3571..4200,1..75))
                     /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(3648..3671)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3846..3865)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4115..4133
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
ORIGIN
        1 ggagccctcc atgtgcacct tgaagcgcat gaactccttg atgatggcca tgttgtcctc
       61 ctcgcccttg ctcacagggc cgggattctc ttccacgtca ccgcatgtca gcagactgcc
      121 tctgccctcg gactagagac cgggcccgag cttaagactg gccgtcgttt tacaacgtcg
      181 tgactgggaa aacatccatg ctagcgttaa cgcgagagta gggaactgcc aggcatcaaa
      241 taaaacgaaa ggctcagtcg gaagactggg cctttcgttt tatctgttgt ttgtcggtga
      301 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgtg aagcaacggc
      361 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaact aagcagaagg
      421 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tcctggctag cggtacgcgt
      481 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
      541 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
      601 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
      661 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
      721 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
      781 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
      841 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
      901 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
      961 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     1021 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     1081 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     1141 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     1201 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     1261 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     1321 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     1381 tacggggtct gacgctcagt ggaacgacgc gtaactcacg ttaagggatt ttggtcatgg
     1441 gtggctcgag ggttatttgc cgactacctt ggtgatctcg cctttcacgt agtggacaaa
     1501 ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct tcttgtccaa gataagcctg
     1561 tctagcttca agtatgacgg gctgatactg ggccggcagg cgctccattg cccagtcggc
     1621 agcgacatcc ttcggcgcga ttttgccggt tactgcgctg taccaaatgc gggacaacgt
     1681 aagcactaca tttcgctcat cgccagccca gtcgggcggc gagttccata gcgttaaggt
     1741 ttcatttagc gcctcaaata gatcctgttc aggaaccgga tcaaagagtt cctccgccgc
     1801 tggacctacc aaggcaacgc tatgttctct tgcttttgtc agcaagatag ccagatcaat
     1861 gtcgatcgtg gctggctcga agatacctgc aagaatgtca ttgcgctgcc attctccaaa
     1921 ttgcagttcg cgcttagctg gataacgcca cggaatgatg tcgtcgtgca caacaatggt
     1981 gacttctaca gcgcggagaa tctcgctctc tccaggggaa gccgaagttt ccaaaaggtc
     2041 gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg gtcaccgtaa ccagcaaatc
     2101 aatatcactg tgtggcttca ggccgccatc cactgcggag ccgtacaaat gtacggccag
     2161 caacgtcggt tcgagatggc gctcgatgac gccaactacc tctgatagtt gagtcgatac
     2221 ttcggcgatc accgcttccc tcataatgtt taactttgtt ttagggcgac tgccctgctg
     2281 cgtaacatcg ttgctgctcc ataacatcaa acatcgaccc acggcgtaac gcgcttgctg
     2341 cttggatgcc cgaggcatag actgtacccc aaaaaaacag tcataacaag ccatgaaaac
     2401 cgccactgcg ccgttaccac cgctgcgttc ggtcaaggtt ctggaccagt tgcgtgagcg
     2461 catacgctac ttgcattaca gcttacgaac cgaacaggct tatgtccact gggttcgtgc
     2521 cttcatccgt ttccacggtg tgcgtcaccc ggcaaccttg ggtagcagcg aagtcgaggc
     2581 atttctgtcc tggctggtct agaggtctct tgaggaatta aaaaacctcc cacacctccc
     2641 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     2701 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     2761 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctacgt
     2821 aatacgactc actatagttc tagactcgag ttagatttcg gtaaaaacag tctctgctga
     2881 ccaacgtgat tttggcagct tagcattaaa atcgtcttcg ctatggtaac ccagcggcac
     2941 aatcaccacg ctggtaaagc ccttctcacg cagaccaaac tcctcatcaa gcaccttggc
     3001 atcgaagcct tcaattggca ccgcgtcgat ctccatcgca gaagcaccca atagcagcgt
     3061 ccccacattg aggtaaactt gcttttccat ccagtgatgc gcgtctttca agtcaaagcg
     3121 gtgcatgttg acgaaaaatg aacgaccagc gtgcatgcca ttttttgctt cgacatcggc
     3181 gtagcggcca tctttgtctt cgctttccaa aaggtcgagt aggtacgctt catcaatcgc
     3241 cgttttggcg cagaacacca caacgtgaga agcatccaaa atcttgcgct cattagcagc
     3301 aaaaccacct tgtgttgctt tggcgatgcg cgcttttccc tcttcactgc tcgcgacaat
     3361 aaaatgccaa ggttgcgagt tgacactgga cgcactcatg cggattaact ctttcactgc
     3421 cgcgactttc tcttcaggca atttgcgcga agcatcaaag gctttggtgg agtagcggga
     3481 ttgggcagct tgaacaatag tcataggccc ggggttctcc tccacgtctc cagcctgctt
     3541 cagcaggctg aagttagtag ctccgcttcc cttgtacagc tcgtccatgc cgccggtgga
     3601 gtggcggccc tcggcgcgct cgtactgttc cacgatggtg tagtcctcgt tgtgggaggt
     3661 gatgtccagc ttgatgttga cgttgtaggc gccgggcagc tgcacgggct tcttggcctt
     3721 gtaggtggtc ttgacctcgg cgtcgtagtg gccgccgtcc ttcagcttca gcctctgctt
     3781 gatctcgccc ttcagggcgc cgtcctcggg gtacatccgc tcggaggagg cctcccagcc
     3841 catggtcttc ttctgcatta cggggccgtc ggaggggaag ttggtgccgc gcagcttcac
     3901 cttgtagatg aactcgccgt cctgcaggga ggagtcctgg gtcacggtca ccacgccgcc
     3961 gtcctcgaag ttcatcacgc gctcccactt gaagccctcg gggaaggaca gcttcaagta
     4021 gtcggggatg tcggcggggt gcttcacgta ggccttggag ccgtacatga actgagggga
     4081 caggatgtcc caggcgaagg gcagggggcc gcccttggtc accttcagct tggcggtctg
     4141 ggtgccctcg taggggcggc cctcgccctc gccctcgatc tcgaactcgt ggccgttcac
//
