LOCUS       sequence-394274-        3551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mini circle middle entry plasmid for membrane GFP- pA with flanking 
            BsaI cut sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3551)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3551)
  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            "Unpublished"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_memb-EGFP-pA
FEATURES             Location/Qualifiers
     source          1..3551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(37..54)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(37..53)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(46..68)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      117..203
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      295..322
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(481..498)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(652..1240)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(732..751)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1333..2124)
                     /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    2561..2695
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2598..2617)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2652..2671
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(2725..3438,3439..3441,3442..3444))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSAQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2770..2791)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3119..3138
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3378..3399
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 gcatggtggc gactagagac cgggcccgag cttaagactg gccgtcgttt tacaacgtcg
       61 tgactgggaa aacatccatg ctagcgttaa cgcgagagta gggaactgcc aggcatcaaa
      121 taaaacgaaa ggctcagtcg gaagactggg cctttcgttt tatctgttgt ttgtcggtga
      181 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgtg aagcaacggc
      241 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaact aagcagaagg
      301 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tcctggctag cggtacgcgt
      361 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
      421 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
      481 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
      541 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
      601 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
      661 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
      721 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
      781 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
      841 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
      901 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
      961 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     1021 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     1081 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     1141 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     1201 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     1261 tacggggtct gacgctcagt ggaacgacgc gtaactcacg ttaagggatt ttggtcatgg
     1321 gtggctcgag ggttatttgc cgactacctt ggtgatctcg cctttcacgt agtggacaaa
     1381 ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct tcttgtccaa gataagcctg
     1441 tctagcttca agtatgacgg gctgatactg ggccggcagg cgctccattg cccagtcggc
     1501 agcgacatcc ttcggcgcga ttttgccggt tactgcgctg taccaaatgc gggacaacgt
     1561 aagcactaca tttcgctcat cgccagccca gtcgggcggc gagttccata gcgttaaggt
     1621 ttcatttagc gcctcaaata gatcctgttc aggaaccgga tcaaagagtt cctccgccgc
     1681 tggacctacc aaggcaacgc tatgttctct tgcttttgtc agcaagatag ccagatcaat
     1741 gtcgatcgtg gctggctcga agatacctgc aagaatgtca ttgcgctgcc attctccaaa
     1801 ttgcagttcg cgcttagctg gataacgcca cggaatgatg tcgtcgtgca caacaatggt
     1861 gacttctaca gcgcggagaa tctcgctctc tccaggggaa gccgaagttt ccaaaaggtc
     1921 gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg gtcaccgtaa ccagcaaatc
     1981 aatatcactg tgtggcttca ggccgccatc cactgcggag ccgtacaaat gtacggccag
     2041 caacgtcggt tcgagatggc gctcgatgac gccaactacc tctgatagtt gagtcgatac
     2101 ttcggcgatc accgcttccc tcataatgtt taactttgtt ttagggcgac tgccctgctg
     2161 cgtaacatcg ttgctgctcc ataacatcaa acatcgaccc acggcgtaac gcgcttgctg
     2221 cttggatgcc cgaggcatag actgtacccc aaaaaaacag tcataacaag ccatgaaaac
     2281 cgccactgcg ccgttaccac cgctgcgttc ggtcaaggtt ctggaccagt tgcgtgagcg
     2341 catacgctac ttgcattaca gcttacgaac cgaacaggct tatgtccact gggttcgtgc
     2401 cttcatccgt ttccacggtg tgcgtcaccc ggcaaccttg ggtagcagcg aagtcgaggc
     2461 atttctgtcc tggctggtct agaggtctct tgaggaatta aaaaacctcc cacacctccc
     2521 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     2581 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     2641 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctacgt
     2701 aatacgactc actatagttc tagactactt gtacagctcg tccatgccga gagtgatccc
     2761 ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct
     2821 cagggcggac tgggtgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat
     2881 gggggtgttc tgctggtagt ggtcggcgag ctgcgcgctg ccgtcctcga tgttgtggcg
     2941 gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg gccatgatat agacgttgtg
     3001 gctgttgtag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat
     3061 gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt
     3121 cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat
     3181 ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcactgcac
     3241 gccgtaggtc agggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca
     3301 gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct
     3361 gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa
     3421 cagctcctcg cccttgctca ccataagctt ggtggcgacc ggtgggtccc gggcccgcgg
     3481 taccgtcgac tgcagctcct gatcggcctc ttcattcttc tcaaccggtt tagttcttct
     3541 gatgcagcac a
//
