LOCUS       sequence-482720-        3603 bp    DNA     circular SYN 08-AUG-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; 
            lifeact-mNeongreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3603)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3603)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 8, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: SA-linker-P2A-lifeact-mNeongreen-ORF-2
FEATURES             Location/Qualifiers
     source          1..3603
                     /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             264..320
                     /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             321..371
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     CDS             375..1082
                     /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      627..636
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1112..1246
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1136..1155)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1190..1209
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1683..2474
                     /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      2567..3155
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3056..3075
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3309..3326
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3485..3512)
                     /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 cagcggctct ggggctacaa acttctctct gctgaaacaa gctggcgacg
      301 tggaggagaa ccctggacct atgggcgtgg ccgacctgat caagaagttc gagagcatca
      361 gcaaggagga gggcgtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
      421 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
      481 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
      541 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
      601 accctgacgg gatgtcgcct ttccaggccg ccatggtaga tggctccggc taccaagtcc
      661 atcgaacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
      721 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
      781 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
      841 acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat ggcaagcgct
      901 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
      961 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
     1021 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaagt
     1081 aagctagaac tatagtgagt cgtattacgt agatccagac atgataagat acattgatga
     1141 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     1201 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     1261 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaat tcctcaagag
     1321 acctctagac cagccaggac agaaatgcct cgacttcgct gctacccaag gttgccgggt
     1381 gacgcacacc gtggaaacgg atgaaggcac gaacccagtg gacataagcc tgttcggttc
     1441 gtaagctgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac
     1501 gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgggg
     1561 tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga
     1621 tgttatggag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaaaca
     1681 ttatgaggga agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca
     1741 tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg
     1801 gcggcctgaa gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg
     1861 aaacaacgcg gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga
     1921 gcgagattct ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc
     1981 gttatccagc taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag
     2041 gtatcttcga gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag
     2101 aacatagcgt tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac
     2161 aggatctatt tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg
     2221 ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg
     2281 gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt
     2341 atcagcccgt catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg
     2401 cctcgcgcgc agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg
     2461 tagtcggcaa ataaccctcg agccacccat gaccaaaatc ccttaacgtg agttacgcgt
     2521 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2581 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2641 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2701 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2761 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2821 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2881 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2941 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3001 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3061 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3121 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3181 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3241 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3301 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     3361 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     3421 cgggcagtga gcgcaacgca attaatacgc gtaccgctag ccaggaagag tttgtagaaa
     3481 cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg
     3541 cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg
     3601 cgg
//
