LOCUS       sequence-481984-        4203 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; NTRv2; 
            mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4203)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4203)
  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: linker-T2A-NTRv2-linker-P2A-mNeonGreen ORF2
FEATURES             Location/Qualifiers
     source          1..4203
                     /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             204..257
                     /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             918..974
                     /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             975..1682
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      1227..1236
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1712..1846
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1736..1755)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1790..1809
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2283..3074
                     /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      3167..3755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3656..3675
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3909..3926
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4085..4112)
                     /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 cgccggctct ggggagggca gaggcagtct gctgacatgc ggtgacgtgg
      241 aagagaatcc cggccctatg actattgttc aagctgccca atcccgctac tccaccaaag
      301 cctttgatgc ttcgcgcaaa ttgcctgaag agaaagtcgc ggcagtgaaa gagttaatcc
      361 gcatgagtgc gtccagtgtc aactcgcaac cttggcattt tattgtcgcg agcagtgaag
      421 agggaaaagc gcgcatcgcc aaagcaacac aaggtggttt tgctgctaat gagcgcaaga
      481 ttttggatgc ttctcacgtt gtggtgttct gcgccaaaac ggcgattgat gaagcgtacc
      541 tactcgacct tttggaaagc gaagacaaag atggccgcta cgccgatgtc gaagcaaaaa
      601 atggcatgca cgctggtcgt tcatttttcg tcaacatgca ccgctttgac ttgaaagacg
      661 cgcatcactg gatggaaaag caagtttacc tcaatgtggg gacgctgcta ttgggtgctt
      721 ctgcgatgga gatcgacgcg gtgccaattg aaggcttcga tgccaaggtg cttgatgagg
      781 agtttggtct gcgtgagaag ggctttacca gcgtggtgat tgtgccgctg ggttaccata
      841 gcgaagacga ttttaatgct aagctgccaa aatcacgttg gtcagcagag actgttttta
      901 ccgaaatcgg ctctggggct acaaacttct ctctgctgaa acaagctggc gacgtggagg
      961 agaaccctgg acctgtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
     1021 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
     1081 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
     1141 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
     1201 accctgacgg gatgtcgcct ttccaggccg ccatggtaga tggctccggc taccaagtcc
     1261 atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
     1321 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
     1381 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
     1441 acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat ggcaagcgct
     1501 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
     1561 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
     1621 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaagt
     1681 aagctagaac tatagtgagt cgtattacgt agatccagac atgataagat acattgatga
     1741 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     1801 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     1861 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaat tcctcaagag
     1921 acctctagac cagccaggac agaaatgcct cgacttcgct gctacccaag gttgccgggt
     1981 gacgcacacc gtggaaacgg atgaaggcac gaacccagtg gacataagcc tgttcggttc
     2041 gtaagctgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac
     2101 gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgggg
     2161 tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga
     2221 tgttatggag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaaaca
     2281 ttatgaggga agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca
     2341 tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg
     2401 gcggcctgaa gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg
     2461 aaacaacgcg gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga
     2521 gcgagattct ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc
     2581 gttatccagc taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag
     2641 gtatcttcga gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag
     2701 aacatagcgt tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac
     2761 aggatctatt tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg
     2821 ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg
     2881 gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt
     2941 atcagcccgt catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg
     3001 cctcgcgcgc agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg
     3061 tagtcggcaa ataaccctcg agccacccat gaccaaaatc ccttaacgtg agttacgcgt
     3121 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3181 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3241 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3301 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3361 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3421 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3481 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3541 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3601 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3661 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3721 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3781 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3841 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3901 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     3961 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     4021 cgggcagtga gcgcaacgca attaatacgc gtaccgctag ccaggaagag tttgtagaaa
     4081 cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg
     4141 cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg
     4201 cgg
//
