LOCUS       sequence-482171-        3604 bp    DNA     circular SYN 06-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 3604)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3604)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 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-3
FEATURES             Location/Qualifiers
     source          1..3604
                     /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             265..321
                     /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             322..372
                     /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             376..1083
                     /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      628..637
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1113..1247
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1137..1156)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1191..1210
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1684..2475
                     /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      2568..3156
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3057..3076
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3310..3327
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3486..3513)
                     /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 cagccggctc tggggctaca aacttctctc tgctgaaaca agctggcgac
      301 gtggaggaga accctggacc tatgggcgtg gccgacctga tcaagaagtt cgagagcatc
      361 agcaaggagg agggcgtgag caagggcgag gaggataaca tggcctctct cccagcgaca
      421 catgagttac acatctttgg ctccatcaac ggtgtggact ttgacatggt gggtcagggc
      481 accggcaatc caaatgatgg ttatgaggag ttaaacctga agtccaccaa gggtgacctc
      541 cagttctccc cctggattct ggtccctcat atcgggtatg gcttccatca gtacctgccc
      601 taccctgacg ggatgtcgcc tttccaggcc gccatggtag atggctccgg ctaccaagtc
      661 catcgcacaa tgcagtttga agatggtgcc tcccttactg ttaactaccg ctacacctac
      721 gagggaagcc acatcaaagg agaggcccag gtgaagggga ctggtttccc tgctgacggt
      781 cctgtgatga ccaactcgct gaccgctgcg gactggtgca ggtcgaagaa gacttacccc
      841 aacgacaaaa ccatcatcag tacctttaag tggagttaca ccactggaaa tggcaagcgc
      901 taccggagca ctgcgcggac cacctacacc tttgccaagc caatggcggc taactatctg
      961 aagaaccagc cgatgtacgt gttccgtaag acggagctca agcactccaa gaccgagctc
     1021 aacttcaagg agtggcaaaa ggcctttacc gatgtgatgg gcatggacga gctgtacaag
     1081 taagctagaa ctatagtgag tcgtattacg tagatccaga catgataaga tacattgatg
     1141 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     1201 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     1261 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa ttcctcaaga
     1321 gacctctaga ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg
     1381 tgacgcacac cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt
     1441 cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa
     1501 cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg
     1561 gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg
     1621 atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac
     1681 attatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc
     1741 atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat
     1801 ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat
     1861 gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag
     1921 agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg
     1981 cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca
     2041 ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga
     2101 gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa
     2161 caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg
     2221 gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc
     2281 ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag
     2341 tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg
     2401 gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag
     2461 gtagtcggca aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg
     2521 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2581 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2641 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2701 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2761 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     2821 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     2881 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     2941 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3001 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3061 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3121 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3181 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3241 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3301 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     3361 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     3421 gcgggcagtg agcgcaacgc aattaatacg cgtaccgcta gccaggaaga gtttgtagaa
     3481 acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg
     3541 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
     3601 gcgg
//
