LOCUS       sequence-482164-        3583 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 3583)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3583)
  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-3
FEATURES             Location/Qualifiers
     source          1..3583
                     /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             205..261
                     /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             373..1059
                     /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     771..790
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1092..1226
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1116..1135)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1170..1189
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1663..2454
                     /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      2547..3135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3036..3055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3289..3306
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3465..3492)
                     /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 cgcccggctc tggggctaca aacttctctc tgctgaaaca agctggcgac
      241 gtggaggaga accctggacc tatgctgtgc tgcatcagaa gaactaaacc ggttgagaag
      301 aatgaagagg ccgatcagga gctgcagtcg acggtaccgc gggcccggga cccaccggtc
      361 gccaccaagc ttatggatag caccgaggca gtgatcaagg agttcatgcg gttcaaggtg
      421 cacatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
      481 ccctacgagg gcacccagac cgccaagctg agggtgacca agggtggccc cctgcccttc
      541 tcctgggaca tcctgtcccc tcagttcatg tacggctcca gggccttcac gaagcacccc
      601 gccgacatcc ccgactactg gaagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg
      661 atgaacttcg aggacggcgg cgccgtgtcc gtggcccagg acacctccct ggaggacggc
      721 accctgatct acaaggtgaa gctccgcggc accaacttcc ctcctgacgg ccccgtaatg
      781 cagaagaaga caatgggctg ggaagcatcc accgagcggt tgtaccccga ggacgtcgtg
      841 ctgaagggcg acattaagat ggccctgcgc ctgaaggacg gcggccgcta cctggcggac
      901 ttcaagacca cctacagggc caagaagccc gtgcagatgc ccggcgcctt caacatcgac
      961 cgcaagttgg acatcacatc ccacaacgag gactacaccg tggtggaaca gtacgaacgc
     1021 tccgtggccc gccactccac cggcggctcc ggtggctcct aatctagaac tatagtgagt
     1081 cgtattacgt agatccagac atgataagat acattgatga gtttggacaa accacaacta
     1141 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     1201 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     1261 ttcaggggga ggtgtgggag gttttttaat tcctcaagag acctctagac cagccaggac
     1321 agaaatgcct cgacttcgct gctacccaag gttgccgggt gacgcacacc gtggaaacgg
     1381 atgaaggcac gaacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag
     1441 cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca
     1501 gtggcggttt tcatggcttg ttatgactgt ttttttgggg tacagtctat gcctcgggca
     1561 tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat
     1621 gttacgcagc agggcagtcg ccctaaaaca aagttaaaca ttatgaggga agcggtgatc
     1681 gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg
     1741 acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt
     1801 gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg
     1861 atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta
     1921 gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa
     1981 ctgcaatttg gcgaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg
     2041 atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta
     2101 ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta
     2161 aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta
     2221 gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat
     2281 gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa
     2341 gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg
     2401 gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataaccctcg
     2461 agccacccat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac
     2521 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2581 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2641 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     2701 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2761 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2821 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2881 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2941 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3001 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3061 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3121 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3181 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3241 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3301 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     3361 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     3421 attaatacgc gtaccgctag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca
     3481 ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc
     3541 ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg cgg
//
