LOCUS       sequence-482168-        3643 bp    DNA     circular SYN 06-AUG-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 3643)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3643)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 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-3
FEATURES             Location/Qualifiers
     source          1..3643
                     /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             265..321
                     /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             433..1119
                     /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     831..850
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1152..1286
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1176..1195)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1230..1249
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1723..2514
                     /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      2607..3195
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3096..3115
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3349..3366
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3525..3552)
                     /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 cagccggctc tggggctaca aacttctctc tgctgaaaca agctggcgac
      301 gtggaggaga accctggacc tatgctgtgc tgcatcagaa gaactaaacc ggttgagaag
      361 aatgaagagg ccgatcagga gctgcagtcg acggtaccgc gggcccggga cccaccggtc
      421 gccaccaagc ttatggatag caccgaggca gtgatcaagg agttcatgcg gttcaaggtg
      481 cacatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
      541 ccctacgagg gcacccagac cgccaagctg agggtgacca agggtggccc cctgcccttc
      601 tcctgggaca tcctgtcccc tcagttcatg tacggctcca gggccttcac gaagcacccc
      661 gccgacatcc ccgactactg gaagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg
      721 atgaacttcg aggacggcgg cgccgtgtcc gtggcccagg acacctccct ggaggacggc
      781 accctgatct acaaggtgaa gctccgcggc accaacttcc ctcctgacgg ccccgtaatg
      841 cagaagaaga caatgggctg ggaagcatcc accgagcggt tgtaccccga ggacgtcgtg
      901 ctgaagggcg acattaagat ggccctgcgc ctgaaggacg gcggccgcta cctggcggac
      961 ttcaagacca cctacagggc caagaagccc gtgcagatgc ccggcgcctt caacatcgac
     1021 cgcaagttgg acatcacatc ccacaacgag gactacaccg tggtggaaca gtacgaacgc
     1081 tccgtggccc gccactccac cggcggctcc ggtggctcct aatctagaac tatagtgagt
     1141 cgtattacgt agatccagac atgataagat acattgatga gtttggacaa accacaacta
     1201 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     1261 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     1321 ttcaggggga ggtgtgggag gttttttaat tcctcaagag acctctagac cagccaggac
     1381 agaaatgcct cgacttcgct gctacccaag gttgccgggt gacgcacacc gtggaaacgg
     1441 atgaaggcac gaacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag
     1501 cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca
     1561 gtggcggttt tcatggcttg ttatgactgt ttttttgggg tacagtctat gcctcgggca
     1621 tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat
     1681 gttacgcagc agggcagtcg ccctaaaaca aagttaaaca ttatgaggga agcggtgatc
     1741 gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg
     1801 acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt
     1861 gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg
     1921 atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta
     1981 gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa
     2041 ctgcaatttg gcgaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg
     2101 atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta
     2161 ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta
     2221 aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta
     2281 gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat
     2341 gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa
     2401 gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg
     2461 gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataaccctcg
     2521 agccacccat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac
     2581 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2641 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2701 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     2761 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2821 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2881 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2941 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3001 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3061 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3121 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3181 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3241 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3301 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3361 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     3421 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     3481 attaatacgc gtaccgctag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca
     3541 ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc
     3601 ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg cgg
//
