LOCUS       sequence-484613-        3548 bp    DNA     circular SYN 26-AUG-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; mNeongreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3548)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3548)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 26, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: SA-linker-P2A-mNeongreen-ORF1
FEATURES             Location/Qualifiers
     source          1..3548
                     /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             263..319
                     /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             320..1027
                     /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      572..581
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1057..1191
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1081..1100)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1135..1154
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1628..2419
                     /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      2512..3100
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3001..3020
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3254..3271
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3430..3457)
                     /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 cagggctctg gggctacaaa cttctctctg ctgaaacaag ctggcgacgt
      301 ggaggagaac cctggacctg tgagcaaggg cgaggaggat aacatggcct ctctcccagc
      361 gacacatgag ttacacatct ttggctccat caacggtgtg gactttgaca tggtgggtca
      421 gggcaccggc aatccaaatg atggttatga ggagttaaac ctgaagtcca ccaagggtga
      481 cctccagttc tccccctgga ttctggtccc tcatatcggg tatggcttcc atcagtacct
      541 gccctaccct gacgggatgt cgcctttcca ggccgccatg gtagatggct ccggctacca
      601 agtccatcgc acaatgcagt ttgaagatgg tgcctccctt actgttaact accgctacac
      661 ctacgaggga agccacatca aaggagaggc ccaggtgaag gggactggtt tccctgctga
      721 cggtcctgtg atgaccaact cgctgaccgc tgcggactgg tgcaggtcga agaagactta
      781 ccccaacgac aaaaccatca tcagtacctt taagtggagt tacaccactg gaaatggcaa
      841 gcgctaccgg agcactgcgc ggaccaccta cacctttgcc aagccaatgg cggctaacta
      901 tctgaagaac cagccgatgt acgtgttccg taagacggag ctcaagcact ccaagaccga
      961 gctcaacttc aaggagtggc aaaaggcctt taccgatgtg atgggcatgg acgagctgta
     1021 caagtaagct agaactatag tgagtcgtat tacgtagatc cagacatgat aagatacatt
     1081 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     1141 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     1201 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaattcctc
     1261 aagagacctc tagaccagcc aggacagaaa tgcctcgact tcgctgctac ccaaggttgc
     1321 cgggtgacgc acaccgtgga aacggatgaa ggcacgaacc cagtggacat aagcctgttc
     1381 ggttcgtaag ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac
     1441 cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt
     1501 tggggtacag tctatgcctc gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg
     1561 tttgatgtta tggagcagca acgatgttac gcagcagggc agtcgcccta aaacaaagtt
     1621 aaacattatg agggaagcgg tgatcgccga agtatcgact caactatcag aggtagttgg
     1681 cgtcatcgag cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt
     1741 ggatggcggc ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct
     1801 tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg
     1861 agagagcgag attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc
     1921 gtggcgttat ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct
     1981 tgcaggtatc ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc
     2041 aagagaacat agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc
     2101 tgaacaggat ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga
     2161 ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt
     2221 aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc
     2281 ccagtatcag cccgtcatac ttgaagctag acaggcttat cttggacaag aagaagatcg
     2341 cttggcctcg cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac
     2401 caaggtagtc ggcaaataac cctcgagcca cccatgacca aaatccctta acgtgagtta
     2461 cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2521 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2581 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2641 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2701 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2761 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2821 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2881 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2941 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3001 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3061 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3121 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3181 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3241 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     3301 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     3361 gaaagcgggc agtgagcgca acgcaattaa tacgcgtacc gctagccagg aagagtttgt
     3421 agaaacgcaa aaaggccatc cgtcaggatg gccttctgct tagtttgatg cctggcagtt
     3481 tatggcgggc gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt caaatccgct
     3541 cccggcgg
//
