LOCUS       sequence-482141-        3581 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 3581)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3581)
  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-1
FEATURES             Location/Qualifiers
     source          1..3581
                     /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             203..259
                     /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             371..1057
                     /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     769..788
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1090..1224
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1168..1187
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1661..2452
                     /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      2545..3133
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3034..3053
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3287..3304
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3463..3490)
                     /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 cgcggctctg gggctacaaa cttctctctg ctgaaacaag ctggcgacgt
      241 ggaggagaac cctggaccta tgctgtgctg catcagaaga actaaaccgg ttgagaagaa
      301 tgaagaggcc gatcaggagc tgcagtcgac ggtaccgcgg gcccgggacc caccggtcgc
      361 caccaagctt atggatagca ccgaggcagt gatcaaggag ttcatgcggt tcaaggtgca
      421 catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
      481 ctacgagggc acccagaccg ccaagctgag ggtgaccaag ggtggccccc tgcccttctc
      541 ctgggacatc ctgtcccctc agttcatgta cggctccagg gccttcacga agcaccccgc
      601 cgacatcccc gactactgga agcagtcctt ccccgagggc ttcaagtggg agcgcgtgat
      661 gaacttcgag gacggcggcg ccgtgtccgt ggcccaggac acctccctgg aggacggcac
      721 cctgatctac aaggtgaagc tccgcggcac caacttccct cctgacggcc ccgtaatgca
      781 gaagaagaca atgggctggg aagcatccac cgagcggttg taccccgagg acgtcgtgct
      841 gaagggcgac attaagatgg ccctgcgcct gaaggacggc ggccgctacc tggcggactt
      901 caagaccacc tacagggcca agaagcccgt gcagatgccc ggcgccttca acatcgaccg
      961 caagttggac atcacatccc acaacgagga ctacaccgtg gtggaacagt acgaacgctc
     1021 cgtggcccgc cactccaccg gcggctccgg tggctcctaa tctagaacta tagtgagtcg
     1081 tattacgtag atccagacat gataagatac attgatgagt ttggacagac cacaactaga
     1141 atgcagtgaa agaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     1201 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     1261 cagggggagg tgtgggaggt tttttaattc ctcaagagac ctctagacca gccaggacag
     1321 aaatgcctcg acttcgctgc tacccaaggt tgccgggtga cgcacaccgt ggaaacggat
     1381 gaaggcacga acccagtgga cataagcctg ttcggttcgt aagctgtaat gcaagtagcg
     1441 tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta acggcgcagt
     1501 ggcggttttc atggcttgtt atgactgttt ttttggggta cagtctatgc ctcgggcatc
     1561 caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt
     1621 tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatt atgagggaag cggtgatcgc
     1681 cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac
     1741 gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga
     1801 tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat
     1861 caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga
     1921 agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact
     1981 gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat
     2041 cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg
     2101 tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa
     2161 tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt
     2221 gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt
     2281 cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc
     2341 tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga
     2401 agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat aaccctcgag
     2461 ccacccatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc
     2521 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2581 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2641 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     2701 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2761 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2821 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2881 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2941 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3001 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3061 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3121 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3181 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3241 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3301 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     3361 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     3421 taatacgcgt accgctagcc aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg
     3481 atggccttct gcttagtttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct
     3541 ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg g
//
