LOCUS       sequence-482023-        4200 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; mCherry; 
            NTR-v2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4200)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4200)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: T2A-mCherry-P2A-NTRv2-pA-ORF-2
FEATURES             Location/Qualifiers
     source          1..4200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             45..98
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             99..803
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(241..259)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     509..528
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     703..726
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             813..869
                     /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"
     polyA_signal    1559..1693
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1583..1602)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1637..1656
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2130..2921
                     /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      3014..3602
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3503..3522
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3756..3773
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3932..3959)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4051..4137)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(4186..4200,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4200,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc ccggtctctt cgccgagggc agaggcagtc
       61 tgctgacatg cggtgacgtg gaagagaatc ccggccctgt gagcaagggc gaggaggaca
      121 acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg
      181 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
      241 ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc
      301 agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga
      361 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg
      421 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc
      481 tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg
      541 aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga
      601 ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc tacaaggcca
      661 agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagctggac atcacctccc
      721 acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg
      781 gcggcatgga cgagctgtac aagggaagcg gagctactaa cttcagcctg ctgaagcagg
      841 ctggagacgt ggaggagaac cccgggccta tgactattgt tcaagctgcc caatcccgct
      901 actccaccaa agcctttgat gcttcgcgca aattgcctga agagaaagtc gcggcagtga
      961 aagagttaat ccgcatgagt gcgtccagtg tcaactcgca accttggcat tttattgtcg
     1021 cgagcagtga agagggaaaa gcgcgcatcg ccaaagcaac acaaggtggt tttgctgcta
     1081 atgagcgcaa gattttggat gcttctcacg ttgtggtgtt ctgcgccaaa acggcgattg
     1141 atgaagcgta cctactcgac cttttggaaa gcgaagacaa agatggccgc tacgccgatg
     1201 tcgaagcaaa aaatggcatg cacgctggtc gttcattttt cgtcaacatg caccgctttg
     1261 acttgaaaga cgcgcatcac tggatggaaa agcaagttta cctcaatgtg gggacgctgc
     1321 tattgggtgc ttctgcgatg gagatcgacg cggtgccaat tgaaggcttc gatgccaagg
     1381 tgcttgatga ggagtttggt ctgcgtgaga agggctttac cagcgtggtg attgtgccgc
     1441 tgggttacca tagcgaagac gattttaatg ctaagctgcc aaaatcacgt tggtcagcag
     1501 agactgtttt taccgaaatc taactcgagt ctagaactat agtgagtcgt attacgtaga
     1561 tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     1621 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     1681 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     1741 gtgggaggtt ttttaattcc tcaagagacc tctagaccag ccaggacaga aatgcctcga
     1801 cttcgctgct acccaaggtt gccgggtgac gcacaccgtg gaaacggatg aaggcacgaa
     1861 cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt atgcgctcac
     1921 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca
     1981 tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag
     2041 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg
     2101 gcagtcgccc taaaacaaag ttaaacatta tgagggaagc ggtgatcgcc gaagtatcga
     2161 ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg
     2221 tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc
     2281 tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt
     2341 tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg
     2401 ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag
     2461 aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc
     2521 tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg
     2581 aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa
     2641 cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt
     2701 cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact
     2761 gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt
     2821 atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc
     2881 actacgtgaa aggcgagatc accaaggtag tcggcaaata accctcgagc cacccatgac
     2941 caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga
     3001 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3061 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3121 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     3181 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3241 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3301 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3361 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3421 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3481 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3541 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3601 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3661 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3721 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3781 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     3841 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatacgcgta
     3901 ccgctagcca ggaagagttt gtagaaacgc aaaaaggcca tccgtcagga tggccttctg
     3961 cttagtttga tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg
     4021 cttcacaacg ttcaaatccg ctcccggcgg atttgtccta ctcaggagag cgttcaccga
     4081 caaacaacag ataaaacgaa aggcccagtc ttccgactga gcctttcgtt ttatttgatg
     4141 cctggcagtt ccctactctc gcgttaacgc tagcatggat gttttcccag tcacgacgtt
//
