LOCUS       sequence-482157-        3641 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 3641)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3641)
  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: SA-linker-P2A-mem-mScarlet-ORF-1
FEATURES             Location/Qualifiers
     source          1..3641
                     /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             263..319
                     /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             431..1117
                     /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     829..848
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1150..1284
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1228..1247
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1721..2512
                     /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      2605..3193
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3094..3113
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3347..3364
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3523..3550)
                     /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 cgccactaat ccggagtgtg tgtgtgtttc tgacgtgtct ctctcttccc
      241 tttttttttt cagggctctg gggctacaaa cttctctctg ctgaaacaag ctggcgacgt
      301 ggaggagaac cctggaccta tgctgtgctg catcagaaga actaaaccgg ttgagaagaa
      361 tgaagaggcc gatcaggagc tgcagtcgac ggtaccgcgg gcccgggacc caccggtcgc
      421 caccaagctt atggatagca ccgaggcagt gatcaaggag ttcatgcggt tcaaggtgca
      481 catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
      541 ctacgagggc acccagaccg ccaagctgag ggtgaccaag ggtggccccc tgcccttctc
      601 ctgggacatc ctgtcccctc agttcatgta cggctccagg gccttcacga agcaccccgc
      661 cgacatcccc gactactgga agcagtcctt ccccgagggc ttcaagtggg agcgcgtgat
      721 gaacttcgag gacggcggcg ccgtgtccgt ggcccaggac acctccctgg aggacggcac
      781 cctgatctac aaggtgaagc tccgcggcac caacttccct cctgacggcc ccgtaatgca
      841 gaagaagaca atgggctggg aagcatccac cgagcggttg taccccgagg acgtcgtgct
      901 gaagggcgac attaagatgg ccctgcgcct gaaggacggc ggccgctacc tggcggactt
      961 caagaccacc tacagggcca agaagcccgt gcagatgccc ggcgccttca acatcgaccg
     1021 caagttggac atcacatccc acaacgagga ctacaccgtg gtggaacagt acgaacgctc
     1081 cgtggcccgc cactccaccg gcggctccgg tggctcctaa tctagaacta tagtgagtcg
     1141 tattacgtag atccagacat gataagatac attgatgagt ttggacagac cacaactaga
     1201 atgcagtgaa agaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     1261 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     1321 cagggggagg tgtgggaggt tttttaattc ctcaagagac ctctagacca gccaggacag
     1381 aaatgcctcg acttcgctgc tacccaaggt tgccgggtga cgcacaccgt ggaaacggat
     1441 gaaggcacga acccagtgga cataagcctg ttcggttcgt aagctgtaat gcaagtagcg
     1501 tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta acggcgcagt
     1561 ggcggttttc atggcttgtt atgactgttt ttttggggta cagtctatgc ctcgggcatc
     1621 caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt
     1681 tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatt atgagggaag cggtgatcgc
     1741 cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac
     1801 gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga
     1861 tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat
     1921 caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga
     1981 agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact
     2041 gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat
     2101 cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg
     2161 tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa
     2221 tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt
     2281 gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt
     2341 cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc
     2401 tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga
     2461 agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat aaccctcgag
     2521 ccacccatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc
     2581 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2641 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2701 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     2761 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2821 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2881 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2941 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3001 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3061 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3121 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3181 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3241 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3301 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3361 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     3421 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     3481 taatacgcgt accgctagcc aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg
     3541 atggccttct gcttagtttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct
     3601 ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg g
//
