LOCUS       sequence-394646-        4191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for T2A- BFP- 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 4191)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4191)
  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            "Unpublished"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-2
FEATURES             Location/Qualifiers
     source          1..4191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     134..156
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     148..165
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     149..165
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             193..246
                     /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             247..942
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             952..1008
                     /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    1698..1832
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1722..1741)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1776..1795
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2269..3060
                     /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      3153..3741
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3642..3661
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3895..3912
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4071..4098)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(join(4190..4191,1..85))
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
       61 ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc gttaacgcta
      121 gcatggatgt tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc
      181 ggtctctagt ccgagggcag aggcagtctg ctgacatgcg gtgacgtgga agagaatccc
      241 ggccctagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg
      301 gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag
      361 accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct
      421 actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc
      481 ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg
      541 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc
      601 aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc
      661 tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaacgac
      721 atggccctga agctcgtggg cgggagccat ctgatcgcaa acatcaagac cacatataga
      781 tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg
      841 gaaagaatca aggaggccaa caacgagacg tacgtcgagc agcacgaggt ggcagtggcc
      901 agatactgcg acctccctag caaactgggg cacaagctta atggaagcgg agctactaac
      961 ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ccgggcctat gactattgtt
     1021 caagctgccc aatcccgcta ctccaccaaa gcctttgatg cttcgcgcaa attgcctgaa
     1081 gagaaagtcg cggcagtgaa agagttaatc cgcatgagtg cgtccagtgt caactcgcaa
     1141 ccttggcatt ttattgtcgc gagcagtgaa gagggaaaag cgcgcatcgc caaagcaaca
     1201 caaggtggtt ttgctgctaa tgagcgcaag attttggatg cttctcacgt tgtggtgttc
     1261 tgcgccaaaa cggcgattga tgaagcgtac ctactcgacc ttttggaaag cgaagacaaa
     1321 gatggccgct acgccgatgt cgaagcaaaa aatggcatgc acgctggtcg ttcatttttc
     1381 gtcaacatgc accgctttga cttgaaagac gcgcatcact ggatggaaaa gcaagtttac
     1441 ctcaatgtgg ggacgctgct attgggtgct tctgcgatgg agatcgacgc ggtgccaatt
     1501 gaaggcttcg atgccaaggt gcttgatgag gagtttggtc tgcgtgagaa gggctttacc
     1561 agcgtggtga ttgtgccgct gggttaccat agcgaagacg attttaatgc taagctgcca
     1621 aaatcacgtt ggtcagcaga gactgttttt accgaaatct aactcgagtc tagaactata
     1681 gtgagtcgta ttacgtagat ccagacatga taagatacat tgatgagttt ggacaaacca
     1741 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     1801 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     1861 ttcaggttca gggggaggtg tgggaggttt tttaattcct caagagacct ctagaccagc
     1921 caggacagaa atgcctcgac ttcgctgcta cccaaggttg ccgggtgacg cacaccgtgg
     1981 aaacggatga aggcacgaac ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc
     2041 aagtagcgta tgcgctcacg caactggtcc agaaccttga ccgaacgcag cggtggtaac
     2101 ggcgcagtgg cggttttcat ggcttgttat gactgttttt ttggggtaca gtctatgcct
     2161 cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc
     2221 aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt taaacattat gagggaagcg
     2281 gtgatcgccg aagtatcgac tcaactatca gaggtagttg gcgtcatcga gcgccatctc
     2341 gaaccgacgt tgctggccgt acatttgtac ggctccgcag tggatggcgg cctgaagcca
     2401 cacagtgata ttgatttgct ggttacggtg accgtaaggc ttgatgaaac aacgcggcga
     2461 gctttgatca acgacctttt ggaaacttcg gcttcccctg gagagagcga gattctccgc
     2521 gctgtagaag tcaccattgt tgtgcacgac gacatcattc cgtggcgtta tccagctaag
     2581 cgcgaactgc aatttggaga atggcagcgc aatgacattc ttgcaggtat cttcgagcca
     2641 gccacgatcg acattgatct ggctatcttg ctgacaaaag caagagaaca tagcgttgcc
     2701 ttggtaggtc cagcggcgga ggaactcttt gatccggttc ctgaacagga tctatttgag
     2761 gcgctaaatg aaaccttaac gctatggaac tcgccgcccg actgggctgg cgatgagcga
     2821 aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg
     2881 aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg cccagtatca gcccgtcata
     2941 cttgaagcta gacaggctta tcttggacaa gaagaagatc gcttggcctc gcgcgcagat
     3001 cagttggaag aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa
     3061 ccctcgagcc acccatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg
     3121 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3181 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3241 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     3301 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3361 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3421 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3481 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3541 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3601 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3661 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3721 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3781 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3841 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3901 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     3961 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     4021 aacgcaatta atacgcgtac cgctagccag gaagagtttg tagaaacgca aaaaggccat
     4081 ccgtcaggat ggccttctgc ttagtttgat gcctggcagt ttatggcggg cgtcctgccc
     4141 gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga t
//
