LOCUS       sequence-482037-        8549 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; 3x-FLAG; 
            mNeongreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8549)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 8549)
  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: nAPEX2-V5-zdCas9n-3x-FLAG-GSG-linker-P2A-mNeon...
FEATURES             Location/Qualifiers
     source          1..8549
                     /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             200..220
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             230..295
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             305..361
                     /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             362..1069
                     /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      614..623
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1099..1233
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1123..1142)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1177..1196
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1670..2461
                     /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      2554..3142
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3043..3062
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3296..3313
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3472..3499)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3591..3677)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3726..3748
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3740..3757
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3741..3757
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      3784..3793
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3802..3822
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3829..4575
                     /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             4588..4629
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
ORIGIN
        1 tatcattcat ctgttcactc tgacaaacct gggcgcacct gcagctttca agtacttcga
       61 cactacaatc gatagaaaga ggtacacctc cactaaggag gtgctggacg ctacactgat
      121 ccatcagagt attaccggcc tgtacgaaac aaggattgac ctgtctcagc tgggtggcga
      181 cgaattcagc agggccgacc ccaagaagaa gaggaaggtg ggccgcggag actacaaaga
      241 ccatgacggt gattataaag atcatgacat cgattacaag gatgacgatg acaagggctc
      301 tggggctaca aacttctctc tgctgaaaca agctggcgac gtggaggaga accctggacc
      361 tgtgagcaag ggcgaggagg ataacatggc ctctctccca gcgacacatg agttacacat
      421 ctttggctcc atcaacggtg tggactttga catggtgggt cagggcaccg gcaatccaaa
      481 tgatggttat gaggagttaa acctgaagtc caccaagggt gacctccagt tctccccctg
      541 gattctggtc cctcatatcg ggtatggctt ccatcagtac ctgccctacc ctgacgggat
      601 gtcgcctttc caggccgcca tggtagatgg ctccggctac caagtccatc gcacaatgca
      661 gtttgaagat ggtgcctccc ttactgttaa ctaccgctac acctacgagg gaagccacat
      721 caaaggagag gcccaggtga aggggactgg tttccctgct gacggtcctg tgatgaccaa
      781 ctcgctgacc gctgcggact ggtgcaggtc gaagaagact taccccaacg acaaaaccat
      841 catcagtacc tttaagtgga gttacaccac tggaaatggc aagcgctacc ggagcactgc
      901 gcggaccacc tacacctttg ccaagccaat ggcggctaac tatctgaaga accagccgat
      961 gtacgtgttc cgtaagacgg agctcaagca ctccaagacc gagctcaact tcaaggagtg
     1021 gcaaaaggcc tttaccgatg tgatgggcat ggacgagctg tacaagtaag ctagaactat
     1081 agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc
     1141 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     1201 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     1261 tttcaggttc agggggaggt gtgggaggtt ttttaattcc tcaagagacc tctagaccag
     1321 ccaggacaga aatgcctcga cttcgctgct acccaaggtt gccgggtgac gcacaccgtg
     1381 gaaacggatg aaggcacgaa cccagtggac ataagcctgt tcggttcgta agctgtaatg
     1441 caagtagcgt atgcgctcac gcaactggtc cagaaccttg accgaacgca gcggtggtaa
     1501 cggcgcagtg gcggttttca tggcttgtta tgactgtttt tttggggtac agtctatgcc
     1561 tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag
     1621 caacgatgtt acgcagcagg gcagtcgccc taaaacaaag ttaaacatta tgagggaagc
     1681 ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg agcgccatct
     1741 cgaaccgacg ttgctggccg tacatttgta cggctccgca gtggatggcg gcctgaagcc
     1801 acacagtgat attgatttgc tggttacggt gaccgtaagg cttgatgaaa caacgcggcg
     1861 agctttgatc aacgaccttt tggaaacttc ggcttcccct ggagagagcg agattctccg
     1921 cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa
     1981 gcgcgaactg caatttggag aatggcagcg caatgacatt cttgcaggta tcttcgagcc
     2041 agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac atagcgttgc
     2101 cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg atctatttga
     2161 ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg gcgatgagcg
     2221 aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc
     2281 gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc agcccgtcat
     2341 acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct cgcgcgcaga
     2401 tcagttggaa gaatttgtcc actacgtgaa aggcgagatc accaaggtag tcggcaaata
     2461 accctcgagc cacccatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc
     2521 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2581 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2641 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2701 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2761 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2821 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2881 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2941 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3001 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3061 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3121 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3181 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3241 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3301 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     3361 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     3421 caacgcaatt aatacgcgta ccgctagcca ggaagagttt gtagaaacgc aaaaaggcca
     3481 tccgtcagga tggccttctg cttagtttga tgcctggcag tttatggcgg gcgtcctgcc
     3541 cgccaccctc cgggccgttg cttcacaacg ttcaaatccg ctcccggcgg atttgtccta
     3601 ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc ttccgactga
     3661 gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcgttaacgc tagcatggat
     3721 gttttcccag tcacgacgtt gtaaaacgac ggccagtctt aagctcgggc ccggtctctt
     3781 cgcgccacca tggcttctcc acctaagaag aagagaaagg tgggaagcgg aaagtcttac
     3841 ccaactgtga gtgctgatta ccaggacgcc gttgagaagg cgaagaagaa gctcagaggc
     3901 ttcatcgctg agaagagatg cgctcctcta atgctccgtt tggcattcca ctctgctgga
     3961 acctttgaca agggcacgaa gaccggtgga cccttcggaa ccatcaagca ccctgccgaa
     4021 ctggctcaca gcgctaacaa cggtcttgac atcgctgtta ggcttttgga gccactcaag
     4081 gcggagttcc ctattttgag ctacgccgat ttctaccagt tggctggcgt tgttgccgtt
     4141 gaggtcacgg gtggacctaa ggttccattc caccctggaa gagaggacaa gcctgagcca
     4201 ccaccagagg gtcgcttgcc tgatcccact aagggttctg accatttgag agatgtgttt
     4261 ggcaaagcta tggggcttac tgaccaagat atcgttgctc tatctggggg tcacactatt
     4321 ggagctgcac acaaggagcg ttctggattt gagggtccct ggacctctaa tcctcttatt
     4381 ttcgacaact catacttcac ggagttgttg agtggtgaga aggaaggact ccttcagcta
     4441 ccttctgaca aggctctttt gtctgaccct gtattccgcc ctctcgttga taaatatgca
     4501 gcggacgaag atgccttctt tgctgattac gctgaggctc accaaaagct ttccgagctt
     4561 gggtttgctg atgccgtgcg gggttctggc aagcccatcc ccaaccccct gctgggcctg
     4621 gacagcacca tggataagaa gtatagcatc ggcctggcca ttggaactaa ctccgtgggt
     4681 tgggcagtga ttacagacga ctacaaggtc cctagcaaga aatttaaggt gctgggtaac
     4741 accgacaggc acagcatcaa gaaaaatctg attggagccc tgctgttcgg ttctggagag
     4801 actgccgaag caacacgcct gaaaagaaca gcaagaaggc gctataccag aaggaagaat
     4861 agaatctgtt acctgcagga gattttctct aacgaaatgg ctaaggtgga cgattcattc
     4921 tttcataggc tggaggaaag tttcctggtc gaggaagata agaaacacga gcgccatcct
     4981 atctttggaa acattgtgga cgaggtcgcc tatcacgaaa aatacccaac catctatcat
     5041 ctgcgcaaga aactggctga ctctactgat aaagccgacc tgagactgat ctatctggct
     5101 ctggcccaca tgattaagtt caggggtcat tttctgatcg agggcgatct gaaccccgac
     5161 aattccgatg tggacaagct gttcatccag ctggtccaga tttacaatca gctgtttgag
     5221 gaaaacccta ttaatgcttc cagagtggac gcaaaagcta tcctgtcagc caggctgtcc
     5281 aagtcacgca gactggagaa cctgattgca cagctgcccg gagaaaagag gaacggtctg
     5341 tttggaaatc tgatcgctct gagtctgggc ctgactccta acttcaaaag caattttgat
     5401 ctggctgagg acgccaaact gcagctgtca aaggacacat atgacgatga cctggataac
     5461 ctgctggcac agatcggaga tcagtacgct gacctgttcc tggctgccaa aaatctgtcc
     5521 gacgcaatcc tgctgtcaga tattctgaga gtgaacagcg agattacaaa agctcctctg
     5581 agtgccagca tgatcaagag atatgacgag caccatcagg atctgaccct gctgaaggct
     5641 ctggtcaggc agcagctgcc agagaagtac aaggaaattt tctttgatca gtccaagaac
     5701 ggctacgccg gttatatcga cggaggcgca tcacaggagg aattctacaa gtttatcaaa
     5761 cctattctgg agaagatgga cggaactgag gaactgctgg tgaaactgaa tagagaggac
     5821 ctgctgagga agcagcgcac atttgataac ggttccatcc cacaccagat tcatctggga
     5881 gagctgcacg ctatcctgag gcgccaggaa gacttctacc cctttctgaa agataaccgc
     5941 gagaagatcg aaaaaattct gaccttcaga atcccttact atgtgggtcc actggctcgc
     6001 ggaaacagca gatttgcctg gatgactcgc aaatccgagg aaaccattac tccttggaac
     6061 ttcgaggaag tggtcgataa gggcgcctct gcacagtcct tcatcgagag aatgactaat
     6121 tttgacaaaa acctgcccaa tgagaaagtg ctgcctaagc actccctgct gtacgagtat
     6181 ttcactgtct ataacgaact gacaaaggtg aaatacgtca ccgagggcat gagaaagcca
     6241 gccttcctgt caggagagca gaagaaagca atcgtggatc tgctgtttaa aaccaacagg
     6301 aaagtgactg tcaagcagct gaaggaggac tacttcaaga aaattgaatg cttcgattcc
     6361 gtggagatca gcggagtcga agacagattt aacgcaagcc tgggcgctta ccacgatctg
     6421 ctgaagatca ttaaggataa agacttcctg gacaacgagg aaaatgagga tatcctggaa
     6481 gacattgtgc tgacactgac cctgtttgag gacagaggaa tgatcgagga aagactgaaa
     6541 acctatgctc atctgttcga tgacaaggtg atgaaacagc tgaagagaag gcgctacact
     6601 ggctggggta gactgagcag gaagctgatc aacggcatta gggataaaca gtcaggaaag
     6661 acaatcctgg actttctgaa aagtgatggc ttcgccaacc gcaattttat gcagctgatt
     6721 cacgatgaca gtctgacctt caaagaggac atccagaagg ctcaggtgtc tggacagggc
     6781 cactccctgc atgagcagat tgcaaacctg gctggaagcc cagccatcaa gaaaggcatt
     6841 ctgcagacag tgaaaatcgt cgatgagctg gtgaaagtca tgggccataa gcccgaaaac
     6901 atcgtgattg agatggctcg cgaaaatcag acaacccaga agggtcagaa gaacagtaga
     6961 gagaggatga aaagaatcga ggaaggcatt aaggagctgg gtagccagat cctgaaagag
     7021 cacccagtgg aaaacacaca gctgcagaat gagaagctgt atctgtacta tctgcagaat
     7081 ggaagagata tgtacgtgga ccaggagctg gatattaaca ggctgtctga ttacgacgtg
     7141 gatgccatcg tcccccagag tttcatcaaa gatgacagca ttgacaacaa ggtgctgacc
     7201 aggtccgaca aaaacagagg aaaatcagat aatgtcccta gtgaggaagt ggtcaagaaa
     7261 atgaagaact actggagaca gctgctgaat gccaaactga tcactcagag gaagtttgat
     7321 aacctgacaa aagcagagcg cggtggactg tcagaactgg acaaagctgg attcatcaag
     7381 aggcagctgg tggaaacacg ccagatcact aaacacgtcg cacagattct ggatagtcgc
     7441 atgaacacaa agtacgatga gaatgacaaa ctgatcagag aagtgaaggt cattaccctg
     7501 aagagtaaac tggtcagcga ctttaggaaa gatttccagt tttataaggt ccgcgagatt
     7561 aacaattatc accatgccca tgacgcatac ctgaacgccg tggtcggtac cgcactgatc
     7621 aagaaatacc caaaactgga gagcgaattc gtgtacggag actataaggt gtacgatgtc
     7681 agaaaaatga tcgccaagtc cgagcaggaa attggaaaag ctactgccaa gtatttcttt
     7741 tactcaaaca tcatgaattt ctttaagaca gagatcaccc tggccaatgg agaaatccgc
     7801 aaaaggcccc tgattgagac aaacggagag acaggcgaaa tcgtgtggga caaaggcaga
     7861 gattttgcaa ccgtgaggaa ggtcctgagc atgcctcaag tgaatatcgt caagaaaact
     7921 gaggtgcaga caggcggttt ctcaaaagaa agtattctgc caaaacgcaa ctctgataag
     7981 ctgatcgcta gaaagaaaga ctgggaccct aagaagtatg gaggctttga ctctcccact
     8041 gtggcatact ccgtcctggt ggtcgctaag gtggagaagg gcaaaagcaa gaaactgaaa
     8101 tctgtcaagg agctgctggg tatcacaatt atggagagaa gctctttcga gaagaaccca
     8161 atcgattttc tggaggccaa aggttataag gaagtgaaga aagacctgat cattaaactg
     8221 cccaagtaca gtctgtttga gctggaaaac ggcaggaaac gcatgctggc aagcgctgga
     8281 gagctgcaga aaggcaatga actggccctg ccttctaagt acgtgaactt cctgtatctg
     8341 gcaagccact acgagaagct gaaaggatct ccagaggata acgaacagaa acagctgttt
     8401 gtggagcagc acaagcatta tctggacgag atcattgaac agattagcga gttctctaaa
     8461 agagtgatcc tggccgacgc aaatctggat aaggtcctgt ctgcttacaa caaacacaga
     8521 gataagccca tcagggagca ggccgaaaa
//
