LOCUS       sequence-482036-        8540 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; V5-APEX2n; 
            mNeongreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8540)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 8540)
  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: 3x-FLAG-nzdCas9-V5-APEX2n-GSG-linker-P2A-mNeon...
FEATURES             Location/Qualifiers
     source          1..8540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             94..102
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             194..235
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             236..982
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     CDS             1001..1021
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1031..1087
                     /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             1088..1795
                     /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      1340..1349
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1825..1959
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1849..1868)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1903..1922
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2396..3187
                     /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      3280..3868
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3769..3788
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4022..4039
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4198..4225)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4317..4403)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4452..4474
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4466..4483
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4467..4483
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      4510..4519
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4519..4584
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4591..4611
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 tatcattcat ctgttcactc tgacaaacct gggcgcacct gcagctttca agtacttcga
       61 cactacaatc gatagaaaga ggtacacctc cactaaggag gtgctggacg ctacactgat
      121 ccatcagagt attaccggcc tgtacgaaac aaggattgac ctgtctcagc tgggtggcga
      181 cgtgcggggt tctggcaagc ccatccccaa ccccctgctg ggcctggaca gcaccggaaa
      241 gtcttaccca actgtgagtg ctgattacca ggacgccgtt gagaaggcga agaagaagct
      301 cagaggcttc atcgctgaga agagatgcgc tcctctaatg ctccgtttgg cattccactc
      361 tgctggaacc tttgacaagg gcacgaagac cggtggaccc ttcggaacca tcaagcaccc
      421 tgccgaactg gctcacagcg ctaacaacgg tcttgacatc gctgttaggc ttttggagcc
      481 actcaaggcg gagttcccta ttttgagcta cgccgatttc taccagttgg ctggcgttgt
      541 tgccgttgag gtcacgggtg gacctaaggt tccattccac cctggaagag aggacaagcc
      601 tgagccacca ccagagggtc gcttgcctga tcccactaag ggttctgacc atttgagaga
      661 tgtgtttggc aaagctatgg ggcttactga ccaagatatc gttgctctat ctgggggtca
      721 cactattgga gctgcacaca aggagcgttc tggatttgag ggtccctgga cctctaatcc
      781 tcttattttc gacaactcat acttcacgga gttgttgagt ggtgagaagg aaggactcct
      841 tcagctacct tctgacaagg ctcttttgtc tgaccctgta ttccgccctc tcgttgataa
      901 atatgcagcg gacgaagatg ccttctttgc tgattacgct gaggctcacc aaaagctttc
      961 cgagcttggg tttgctgatg ccgaattcag cagggccgac cccaagaaga agaggaaggt
     1021 gggctctggg gctacaaact tctctctgct gaaacaagct ggcgacgtgg aggagaaccc
     1081 tggacctgtg agcaagggcg aggaggataa catggcctct ctcccagcga cacatgagtt
     1141 acacatcttt ggctccatca acggtgtgga ctttgacatg gtgggtcagg gcaccggcaa
     1201 tccaaatgat ggttatgagg agttaaacct gaagtccacc aagggtgacc tccagttctc
     1261 cccctggatt ctggtccctc atatcgggta tggcttccat cagtacctgc cctaccctga
     1321 cgggatgtcg cctttccagg ccgccatggt agatggctcc ggctaccaag tccatcgcac
     1381 aatgcagttt gaagatggtg cctcccttac tgttaactac cgctacacct acgagggaag
     1441 ccacatcaaa ggagaggccc aggtgaaggg gactggtttc cctgctgacg gtcctgtgat
     1501 gaccaactcg ctgaccgctg cggactggtg caggtcgaag aagacttacc ccaacgacaa
     1561 aaccatcatc agtaccttta agtggagtta caccactgga aatggcaagc gctaccggag
     1621 cactgcgcgg accacctaca cctttgccaa gccaatggcg gctaactatc tgaagaacca
     1681 gccgatgtac gtgttccgta agacggagct caagcactcc aagaccgagc tcaacttcaa
     1741 ggagtggcaa aaggccttta ccgatgtgat gggcatggac gagctgtaca agtaagctag
     1801 aactatagtg agtcgtatta cgtagatcca gacatgataa gatacattga tgagtttgga
     1861 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     1921 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     1981 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aattcctcaa gagacctcta
     2041 gaccagccag gacagaaatg cctcgacttc gctgctaccc aaggttgccg ggtgacgcac
     2101 accgtggaaa cggatgaagg cacgaaccca gtggacataa gcctgttcgg ttcgtaagct
     2161 gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga accttgaccg aacgcagcgg
     2221 tggtaacggc gcagtggcgg ttttcatggc ttgttatgac tgtttttttg gggtacagtc
     2281 tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg
     2341 gagcagcaac gatgttacgc agcagggcag tcgccctaaa acaaagttaa acattatgag
     2401 ggaagcggtg atcgccgaag tatcgactca actatcagag gtagttggcg tcatcgagcg
     2461 ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg atggcggcct
     2521 gaagccacac agtgatattg atttgctggt tacggtgacc gtaaggcttg atgaaacaac
     2581 gcggcgagct ttgatcaacg accttttgga aacttcggct tcccctggag agagcgagat
     2641 tctccgcgct gtagaagtca ccattgttgt gcacgacgac atcattccgt ggcgttatcc
     2701 agctaagcgc gaactgcaat ttggagaatg gcagcgcaat gacattcttg caggtatctt
     2761 cgagccagcc acgatcgaca ttgatctggc tatcttgctg acaaaagcaa gagaacatag
     2821 cgttgccttg gtaggtccag cggcggagga actctttgat ccggttcctg aacaggatct
     2881 atttgaggcg ctaaatgaaa ccttaacgct atggaactcg ccgcccgact gggctggcga
     2941 tgagcgaaat gtagtgctta cgttgtcccg catttggtac agcgcagtaa ccggcaaaat
     3001 cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc ctgccggccc agtatcagcc
     3061 cgtcatactt gaagctagac aggcttatct tggacaagaa gaagatcgct tggcctcgcg
     3121 cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg
     3181 caaataaccc tcgagccacc catgaccaaa atcccttaac gtgagttacg cgtcgttcca
     3241 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3301 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3361 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3421 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3481 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3541 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3601 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3661 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3721 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3781 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3841 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3901 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3961 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4021 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     4081 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     4141 tgagcgcaac gcaattaata cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa
     4201 aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt
     4261 cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt
     4321 gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc
     4381 gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcgt taacgctagc
     4441 atggatgttt tcccagtcac gacgttgtaa aacgacggcc agtcttaagc tcgggcccgg
     4501 tctcttcgcg ccaccatgga ctacaaagac catgacggtg attataaaga tcatgacatc
     4561 gattacaagg atgacgatga caagatggcc cccaagaaga agaggaaggt gggccgcgga
     4621 atggataaga agtatagcat cggcctggcc attggaacta actccgtggg ttgggcagtg
     4681 attacagacg actacaaggt ccctagcaag aaatttaagg tgctgggtaa caccgacagg
     4741 cacagcatca agaaaaatct gattggagcc ctgctgttcg gttctggaga gactgccgaa
     4801 gcaacacgcc tgaaaagaac agcaagaagg cgctatacca gaaggaagaa tagaatctgt
     4861 tacctgcagg agattttctc taacgaaatg gctaaggtgg acgattcatt ctttcatagg
     4921 ctggaggaaa gtttcctggt cgaggaagat aagaaacacg agcgccatcc tatctttgga
     4981 aacattgtgg acgaggtcgc ctatcacgaa aaatacccaa ccatctatca tctgcgcaag
     5041 aaactggctg actctactga taaagccgac ctgagactga tctatctggc tctggcccac
     5101 atgattaagt tcaggggtca ttttctgatc gagggcgatc tgaaccccga caattccgat
     5161 gtggacaagc tgttcatcca gctggtccag atttacaatc agctgtttga ggaaaaccct
     5221 attaatgctt ccagagtgga cgcaaaagct atcctgtcag ccaggctgtc caagtcacgc
     5281 agactggaga acctgattgc acagctgccc ggagaaaaga ggaacggtct gtttggaaat
     5341 ctgatcgctc tgagtctggg cctgactcct aacttcaaaa gcaattttga tctggctgag
     5401 gacgccaaac tgcagctgtc aaaggacaca tatgacgatg acctggataa cctgctggca
     5461 cagatcggag atcagtacgc tgacctgttc ctggctgcca aaaatctgtc cgacgcaatc
     5521 ctgctgtcag atattctgag agtgaacagc gagattacaa aagctcctct gagtgccagc
     5581 atgatcaaga gatatgacga gcaccatcag gatctgaccc tgctgaaggc tctggtcagg
     5641 cagcagctgc cagagaagta caaggaaatt ttctttgatc agtccaagaa cggctacgcc
     5701 ggttatatcg acggaggcgc atcacaggag gaattctaca agtttatcaa acctattctg
     5761 gagaagatgg acggaactga ggaactgctg gtgaaactga atagagagga cctgctgagg
     5821 aagcagcgca catttgataa cggttccatc ccacaccaga ttcatctggg agagctgcac
     5881 gctatcctga ggcgccagga agacttctac ccctttctga aagataaccg cgagaagatc
     5941 gaaaaaattc tgaccttcag aatcccttac tatgtgggtc cactggctcg cggaaacagc
     6001 agatttgcct ggatgactcg caaatccgag gaaaccatta ctccttggaa cttcgaggaa
     6061 gtggtcgata agggcgcctc tgcacagtcc ttcatcgaga gaatgactaa ttttgacaaa
     6121 aacctgccca atgagaaagt gctgcctaag cactccctgc tgtacgagta tttcactgtc
     6181 tataacgaac tgacaaaggt gaaatacgtc accgagggca tgagaaagcc agccttcctg
     6241 tcaggagagc agaagaaagc aatcgtggat ctgctgttta aaaccaacag gaaagtgact
     6301 gtcaagcagc tgaaggagga ctacttcaag aaaattgaat gcttcgattc cgtggagatc
     6361 agcggagtcg aagacagatt taacgcaagc ctgggcgctt accacgatct gctgaagatc
     6421 attaaggata aagacttcct ggacaacgag gaaaatgagg atatcctgga agacattgtg
     6481 ctgacactga ccctgtttga ggacagagga atgatcgagg aaagactgaa aacctatgct
     6541 catctgttcg atgacaaggt gatgaaacag ctgaagagaa ggcgctacac tggctggggt
     6601 agactgagca ggaagctgat caacggcatt agggataaac agtcaggaaa gacaatcctg
     6661 gactttctga aaagtgatgg cttcgccaac cgcaatttta tgcagctgat tcacgatgac
     6721 agtctgacct tcaaagagga catccagaag gctcaggtgt ctggacaggg ccactccctg
     6781 catgagcaga ttgcaaacct ggctggaagc ccagccatca agaaaggcat tctgcagaca
     6841 gtgaaaatcg tcgatgagct ggtgaaagtc atgggccata agcccgaaaa catcgtgatt
     6901 gagatggctc gcgaaaatca gacaacccag aagggtcaga agaacagtag agagaggatg
     6961 aaaagaatcg aggaaggcat taaggagctg ggtagccaga tcctgaaaga gcacccagtg
     7021 gaaaacacac agctgcagaa tgagaagctg tatctgtact atctgcagaa tggaagagat
     7081 atgtacgtgg accaggagct ggatattaac aggctgtctg attacgacgt ggatgccatc
     7141 gtcccccaga gtttcatcaa agatgacagc attgacaaca aggtgctgac caggtccgac
     7201 aaaaacagag gaaaatcaga taatgtccct agtgaggaag tggtcaagaa aatgaagaac
     7261 tactggagac agctgctgaa tgccaaactg atcactcaga ggaagtttga taacctgaca
     7321 aaagcagagc gcggtggact gtcagaactg gacaaagctg gattcatcaa gaggcagctg
     7381 gtggaaacac gccagatcac taaacacgtc gcacagattc tggatagtcg catgaacaca
     7441 aagtacgatg agaatgacaa actgatcaga gaagtgaagg tcattaccct gaagagtaaa
     7501 ctggtcagcg actttaggaa agatttccag ttttataagg tccgcgagat taacaattat
     7561 caccatgccc atgacgcata cctgaacgcc gtggtcggta ccgcactgat caagaaatac
     7621 ccaaaactgg agagcgaatt cgtgtacgga gactataagg tgtacgatgt cagaaaaatg
     7681 atcgccaagt ccgagcagga aattggaaaa gctactgcca agtatttctt ttactcaaac
     7741 atcatgaatt tctttaagac agagatcacc ctggccaatg gagaaatccg caaaaggccc
     7801 ctgattgaga caaacggaga gacaggcgaa atcgtgtggg acaaaggcag agattttgca
     7861 accgtgagga aggtcctgag catgcctcaa gtgaatatcg tcaagaaaac tgaggtgcag
     7921 acaggcggtt tctcaaaaga aagtattctg ccaaaacgca actctgataa gctgatcgct
     7981 agaaagaaag actgggaccc taagaagtat ggaggctttg actctcccac tgtggcatac
     8041 tccgtcctgg tggtcgctaa ggtggagaag ggcaaaagca agaaactgaa atctgtcaag
     8101 gagctgctgg gtatcacaat tatggagaga agctctttcg agaagaaccc aatcgatttt
     8161 ctggaggcca aaggttataa ggaagtgaag aaagacctga tcattaaact gcccaagtac
     8221 agtctgtttg agctggaaaa cggcaggaaa cgcatgctgg caagcgctgg agagctgcag
     8281 aaaggcaatg aactggccct gccttctaag tacgtgaact tcctgtatct ggcaagccac
     8341 tacgagaagc tgaaaggatc tccagaggat aacgaacaga aacagctgtt tgtggagcag
     8401 cacaagcatt atctggacga gatcattgaa cagattagcg agttctctaa aagagtgatc
     8461 ctggccgacg caaatctgga taaggtcctg tctgcttaca acaaacacag agataagccc
     8521 atcagggagc aggccgaaaa
//
