LOCUS       sequence-482025-        4199 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 4199)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4199)
  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-1
FEATURES             Location/Qualifiers
     source          1..4199
                     /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             44..97
                     /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             98..802
                     /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(240..258)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     508..527
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     702..725
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             812..868
                     /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    1558..1692
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1582..1601)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1636..1655
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2129..2920
                     /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      3013..3601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3502..3521
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3755..3772
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3931..3958)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4050..4136)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(4185..4199,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4199,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc ccggtctctt cgcgagggca gaggcagtct
       61 gctgacatgc ggtgacgtgg aagagaatcc cggccctgtg agcaagggcg aggaggacaa
      121 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg
      181 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
      241 caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccctca
      301 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
      361 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
      421 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
      481 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
      541 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
      601 gctgaagctg aaggacggcg gccactacga cgccgaggtc aagaccacct acaaggccaa
      661 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagctggaca tcacctccca
      721 caacgaggac tacaccatcg tggaacagta cgagcgcgcc gagggccgcc actccaccgg
      781 cggcatggac gagctgtaca agggaagcgg agctactaac ttcagcctgc tgaagcaggc
      841 tggagacgtg gaggagaacc ccgggcctat gactattgtt caagctgccc aatcccgcta
      901 ctccaccaaa gcctttgatg cttcgcgcaa attgcctgaa gagaaagtcg cggcagtgaa
      961 agagttaatc cgcatgagtg cgtccagtgt caactcgcaa ccttggcatt ttattgtcgc
     1021 gagcagtgaa gagggaaaag cgcgcatcgc caaagcaaca caaggtggtt ttgctgctaa
     1081 tgagcgcaag attttggatg cttctcacgt tgtggtgttc tgcgccaaaa cggcgattga
     1141 tgaagcgtac ctactcgacc ttttggaaag cgaagacaaa gatggccgct acgccgatgt
     1201 cgaagcaaaa aatggcatgc acgctggtcg ttcatttttc gtcaacatgc accgctttga
     1261 cttgaaagac gcgcatcact ggatggaaaa gcaagtttac ctcaatgtgg ggacgctgct
     1321 attgggtgct tctgcgatgg agatcgacgc ggtgccaatt gaaggcttcg atgccaaggt
     1381 gcttgatgag gagtttggtc tgcgtgagaa gggctttacc agcgtggtga ttgtgccgct
     1441 gggttaccat agcgaagacg attttaatgc taagctgcca aaatcacgtt ggtcagcaga
     1501 gactgttttt accgaaatct aactcgagtc tagaactata gtgagtcgta ttacgtagat
     1561 ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     1621 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     1681 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     1741 tgggaggttt tttaattcct caagagacct ctagaccagc caggacagaa atgcctcgac
     1801 ttcgctgcta cccaaggttg ccgggtgacg cacaccgtgg aaacggatga aggcacgaac
     1861 ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg
     1921 caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat
     1981 ggcttgttat gactgttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc
     2041 gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg
     2101 cagtcgccct aaaacaaagt taaacattat gagggaagcg gtgatcgccg aagtatcgac
     2161 tcaactatca gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt
     2221 acatttgtac ggctccgcag tggatggcgg cctgaagcca cacagtgata ttgatttgct
     2281 ggttacggtg accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt
     2341 ggaaacttcg gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt
     2401 tgtgcacgac gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga
     2461 atggcagcgc aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct
     2521 ggctatcttg ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga
     2581 ggaactcttt gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac
     2641 gctatggaac tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc
     2701 ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg
     2761 ggcaatggag cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta
     2821 tcttggacaa gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca
     2881 ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa ccctcgagcc acccatgacc
     2941 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     3001 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3061 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3121 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     3181 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3241 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3301 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3361 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3421 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3481 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3541 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3601 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3661 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3721 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3781 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3841 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac
     3901 cgctagccag gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc
     3961 ttagtttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc
     4021 ttcacaacgt tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac
     4081 aaacaacaga taaaacgaaa ggcccagtct tccgactgag cctttcgttt tatttgatgc
     4141 ctggcagttc cctactctcg cgttaacgct agcatggatg ttttcccagt cacgacgtt
//
