LOCUS       sequence-482140-        3582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; mScarlet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3582)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3582)
  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"
COMMENT     Sequence Label: linker-P2A-mem-mScarlet-ORF-2
FEATURES             Location/Qualifiers
     source          1..3582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(1..87)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     136..158
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     150..167
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     151..167
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             204..260
                     /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             372..1058
                     /codon_start=1
                     /product="bright cysteine-free monomeric red fluorescent 
                     protein with fast and complete maturation (Gadella et al., 
                     2023)"
                     /label=mScarlet3
                     /translation="MDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LRVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGA
                     VSVAQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKM
                     ALRLKDGGRYLADFKTTYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHS
                     TGGSGGS"
     primer_bind     770..789
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1091..1225
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1115..1134)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1169..1188
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1662..2453
                     /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      2546..3134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3035..3054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3288..3305
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3464..3491)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
       61 ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcgttaacgc
      121 tagcatggat gttttcccag tcacgacgtt gtaaaacgac ggccagtctt aagctcgggc
      181 ccggtctctt cgccggctct ggggctacaa acttctctct gctgaaacaa gctggcgacg
      241 tggaggagaa ccctggacct atgctgtgct gcatcagaag aactaaaccg gttgagaaga
      301 atgaagaggc cgatcaggag ctgcagtcga cggtaccgcg ggcccgggac ccaccggtcg
      361 ccaccaagct tatggatagc accgaggcag tgatcaagga gttcatgcgg ttcaaggtgc
      421 acatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
      481 cctacgaggg cacccagacc gccaagctga gggtgaccaa gggtggcccc ctgcccttct
      541 cctgggacat cctgtcccct cagttcatgt acggctccag ggccttcacg aagcaccccg
      601 ccgacatccc cgactactgg aagcagtcct tccccgaggg cttcaagtgg gagcgcgtga
      661 tgaacttcga ggacggcggc gccgtgtccg tggcccagga cacctccctg gaggacggca
      721 ccctgatcta caaggtgaag ctccgcggca ccaacttccc tcctgacggc cccgtaatgc
      781 agaagaagac aatgggctgg gaagcatcca ccgagcggtt gtaccccgag gacgtcgtgc
      841 tgaagggcga cattaagatg gccctgcgcc tgaaggacgg cggccgctac ctggcggact
      901 tcaagaccac ctacagggcc aagaagcccg tgcagatgcc cggcgccttc aacatcgacc
      961 gcaagttgga catcacatcc cacaacgagg actacaccgt ggtggaacag tacgaacgct
     1021 ccgtggcccg ccactccacc ggcggctccg gtggctccta atctagaact atagtgagtc
     1081 gtattacgta gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag
     1141 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     1201 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     1261 tcagggggag gtgtgggagg ttttttaatt cctcaagaga cctctagacc agccaggaca
     1321 gaaatgcctc gacttcgctg ctacccaagg ttgccgggtg acgcacaccg tggaaacgga
     1381 tgaaggcacg aacccagtgg acataagcct gttcggttcg taagctgtaa tgcaagtagc
     1441 gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt aacggcgcag
     1501 tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg cctcgggcat
     1561 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
     1621 ttacgcagca gggcagtcgc cctaaaacaa agttaaacat tatgagggaa gcggtgatcg
     1681 ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga
     1741 cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg
     1801 atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga
     1861 tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag
     1921 aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac
     1981 tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga
     2041 tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag
     2101 gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa
     2161 atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag
     2221 tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg
     2281 tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag
     2341 ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg
     2401 aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa taaccctcga
     2461 gccacccatg accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc
     2521 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2581 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2641 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2701 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2761 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2821 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2881 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2941 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3001 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3061 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3121 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3181 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3241 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3301 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     3361 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     3421 ttaatacgcg taccgctagc caggaagagt ttgtagaaac gcaaaaaggc catccgtcag
     3481 gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc
     3541 tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc gg
//
