LOCUS       sequence-482042-        4154 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 4154)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4154)
  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: mCherry-P2A-NTRv2-pA
FEATURES             Location/Qualifiers
     source          1..4154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      44..53
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             50..757
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(195..213)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     463..482
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     657..680
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             767..823
                     /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    1513..1647
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1537..1556)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1591..1610
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2084..2875
                     /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      2968..3556
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3457..3476
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3710..3727
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3886..3913)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4005..4091)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(4140..4154,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4154,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc ccggtctctt cgcgccacca tggtgagcaa
       61 gggcgaggag gacaacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga
      121 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
      181 gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga
      241 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
      301 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
      361 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
      421 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
      481 gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
      541 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgccg aggtcaagac
      601 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagct
      661 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc gcgccgaggg
      721 ccgccactcc accggcggca tggacgagct gtacaaggga agcggagcta ctaacttcag
      781 cctgctgaag caggctggag acgtggagga gaaccccggg cctatgacta ttgttcaagc
      841 tgcccaatcc cgctactcca ccaaagcctt tgatgcttcg cgcaaattgc ctgaagagaa
      901 agtcgcggca gtgaaagagt taatccgcat gagtgcgtcc agtgtcaact cgcaaccttg
      961 gcattttatt gtcgcgagca gtgaagaggg aaaagcgcgc atcgccaaag caacacaagg
     1021 tggttttgct gctaatgagc gcaagatttt ggatgcttct cacgttgtgg tgttctgcgc
     1081 caaaacggcg attgatgaag cgtacctact cgaccttttg gaaagcgaag acaaagatgg
     1141 ccgctacgcc gatgtcgaag caaaaaatgg catgcacgct ggtcgttcat ttttcgtcaa
     1201 catgcaccgc tttgacttga aagacgcgca tcactggatg gaaaagcaag tttacctcaa
     1261 tgtggggacg ctgctattgg gtgcttctgc gatggagatc gacgcggtgc caattgaagg
     1321 cttcgatgcc aaggtgcttg atgaggagtt tggtctgcgt gagaagggct ttaccagcgt
     1381 ggtgattgtg ccgctgggtt accatagcga agacgatttt aatgctaagc tgccaaaatc
     1441 acgttggtca gcagagactg tttttaccga aatctaactc gagtctagaa ctatagtgag
     1501 tcgtattacg tagatccaga catgataaga tacattgatg agtttggaca aaccacaact
     1561 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     1621 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     1681 gttcaggggg aggtgtggga ggttttttaa ttcctcaaga gacctctaga ccagccagga
     1741 cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg tgacgcacac cgtggaaacg
     1801 gatgaaggca cgaacccagt ggacataagc ctgttcggtt cgtaagctgt aatgcaagta
     1861 gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg gtaacggcgc
     1921 agtggcggtt ttcatggctt gttatgactg tttttttggg gtacagtcta tgcctcgggc
     1981 atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga gcagcaacga
     2041 tgttacgcag cagggcagtc gccctaaaac aaagttaaac attatgaggg aagcggtgat
     2101 cgccgaagta tcgactcaac tatcagaggt agttggcgtc atcgagcgcc atctcgaacc
     2161 gacgttgctg gccgtacatt tgtacggctc cgcagtggat ggcggcctga agccacacag
     2221 tgatattgat ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc ggcgagcttt
     2281 gatcaacgac cttttggaaa cttcggcttc ccctggagag agcgagattc tccgcgctgt
     2341 agaagtcacc attgttgtgc acgacgacat cattccgtgg cgttatccag ctaagcgcga
     2401 actgcaattt ggagaatggc agcgcaatga cattcttgca ggtatcttcg agccagccac
     2461 gatcgacatt gatctggcta tcttgctgac aaaagcaaga gaacatagcg ttgccttggt
     2521 aggtccagcg gcggaggaac tctttgatcc ggttcctgaa caggatctat ttgaggcgct
     2581 aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg gctggcgatg agcgaaatgt
     2641 agtgcttacg ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg cgccgaagga
     2701 tgtcgctgcc gactgggcaa tggagcgcct gccggcccag tatcagcccg tcatacttga
     2761 agctagacag gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg cagatcagtt
     2821 ggaagaattt gtccactacg tgaaaggcga gatcaccaag gtagtcggca aataaccctc
     2881 gagccaccca tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga
     2941 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3001 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3061 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     3121 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3181 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3241 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3301 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3361 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3421 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3481 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3541 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3601 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3661 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3721 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     3781 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     3841 aattaatacg cgtaccgcta gccaggaaga gtttgtagaa acgcaaaaag gccatccgtc
     3901 aggatggcct tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac
     3961 cctccgggcc gttgcttcac aacgttcaaa tccgctcccg gcggatttgt cctactcagg
     4021 agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtcttccga ctgagccttt
     4081 cgttttattt gatgcctggc agttccctac tctcgcgtta acgctagcat ggatgttttc
     4141 ccagtcacga cgtt
//
