LOCUS       sequence-482166-        7670 bp    DNA     circular SYN 06-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 7670)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 7670)
  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: nzCas9n-GSG-linker-P2A-mNeongreen
FEATURES             Location/Qualifiers
     source          1..7670
                     /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"
     regulatory      194..203
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             212..232
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             239..4339
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes MGAS15252 Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRL
                     ENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             4252..4260
                     /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             4355..4375
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4385..4441
                     /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             4442..5149
                     /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      4694..4703
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    5179..5313
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5203..5222)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5257..5276
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             5750..6541
                     /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      6634..7222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7123..7142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7376..7393
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(7552..7579)
                     /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 cgcgccacca tggcttctcc acctaagaag aagagaaagg tgggaagcat
      241 ggataagaag tatagcatcg gcctggatat tggaactaac tccgtgggtt gggcagtgat
      301 tacagacgac tacaaggtcc ctagcaagaa atttaaggtg ctgggtaaca ccgacaggca
      361 cagcatcaag aaaaatctga ttggagccct gctgttcggt tctggagaga ctgccgaagc
      421 aacacgcctg aaaagaacag caagaaggcg ctataccaga aggaagaata gaatctgtta
      481 cctgcaggag attttctcta acgaaatggc taaggtggac gattcattct ttcataggct
      541 ggaggaaagt ttcctggtcg aggaagataa gaaacacgag cgccatccta tctttggaaa
      601 cattgtggac gaggtcgcct atcacgaaaa atacccaacc atctatcatc tgcgcaagaa
      661 actggctgac tctactgata aagccgacct gagactgatc tatctggctc tggcccacat
      721 gattaagttc aggggtcatt ttctgatcga gggcgatctg aaccccgaca attccgatgt
      781 ggacaagctg ttcatccagc tggtccagat ttacaatcag ctgtttgagg aaaaccctat
      841 taatgcttcc agagtggacg caaaagctat cctgtcagcc aggctgtcca agtcacgcag
      901 actggagaac ctgattgcac agctgcccgg agaaaagagg aacggtctgt ttggaaatct
      961 gatcgctctg agtctgggcc tgactcctaa cttcaaaagc aattttgatc tggctgagga
     1021 cgccaaactg cagctgtcaa aggacacata tgacgatgac ctggataacc tgctggcaca
     1081 gatcggagat cagtacgctg acctgttcct ggctgccaaa aatctgtccg acgcaatcct
     1141 gctgtcagat attctgagag tgaacagcga gattacaaaa gctcctctga gtgccagcat
     1201 gatcaagaga tatgacgagc accatcagga tctgaccctg ctgaaggctc tggtcaggca
     1261 gcagctgcca gagaagtaca aggaaatttt ctttgatcag tccaagaacg gctacgccgg
     1321 ttatatcgac ggaggcgcat cacaggagga attctacaag tttatcaaac ctattctgga
     1381 gaagatggac ggaactgagg aactgctggt gaaactgaat agagaggacc tgctgaggaa
     1441 gcagcgcaca tttgataacg gttccatccc acaccagatt catctgggag agctgcacgc
     1501 tatcctgagg cgccaggaag acttctaccc ctttctgaaa gataaccgcg agaagatcga
     1561 aaaaattctg accttcagaa tcccttacta tgtgggtcca ctggctcgcg gaaacagcag
     1621 atttgcctgg atgactcgca aatccgagga aaccattact ccttggaact tcgaggaagt
     1681 ggtcgataag ggcgcctctg cacagtcctt catcgagaga atgactaatt ttgacaaaaa
     1741 cctgcccaat gagaaagtgc tgcctaagca ctccctgctg tacgagtatt tcactgtcta
     1801 taacgaactg acaaaggtga aatacgtcac cgagggcatg agaaagccag ccttcctgtc
     1861 aggagagcag aagaaagcaa tcgtggatct gctgtttaaa accaacagga aagtgactgt
     1921 caagcagctg aaggaggact acttcaagaa aattgaatgc ttcgattccg tggagatcag
     1981 cggagtcgaa gacagattta acgcaagcct gggcgcttac cacgatctgc tgaagatcat
     2041 taaggataaa gacttcctgg acaacgagga aaatgaggat atcctggaag acattgtgct
     2101 gacactgacc ctgtttgagg acagaggaat gatcgaggaa agactgaaaa cctatgctca
     2161 tctgttcgat gacaaggtga tgaaacagct gaagagaagg cgctacactg gctggggtag
     2221 actgagcagg aagctgatca acggcattag ggataaacag tcaggaaaga caatcctgga
     2281 ctttctgaaa agtgatggct tcgccaaccg caattttatg cagctgattc acgatgacag
     2341 tctgaccttc aaagaggaca tccagaaggc tcaggtgtct ggacagggcc actccctgca
     2401 tgagcagatt gcaaacctgg ctggaagccc agccatcaag aaaggcattc tgcagacagt
     2461 gaaaatcgtc gatgagctgg tgaaagtcat gggccataag cccgaaaaca tcgtgattga
     2521 gatggctcgc gaaaatcaga caacccagaa gggtcagaag aacagtagag agaggatgaa
     2581 aagaatcgag gaaggcatta aggagctggg tagccagatc ctgaaagagc acccagtgga
     2641 aaacacacag ctgcagaatg agaagctgta tctgtactat ctgcagaatg gaagagatat
     2701 gtacgtggac caggagctgg atattaacag gctgtctgat tacgacgtgg atcatatcgt
     2761 cccccagagt ttcatcaaag atgacagcat tgacaacaag gtgctgacca ggtccgacaa
     2821 aaacagagga aaatcagata atgtccctag tgaggaagtg gtcaagaaaa tgaagaacta
     2881 ctggagacag ctgctgaatg ccaaactgat cactcagagg aagtttgata acctgacaaa
     2941 agcagagcgc ggtggactgt cagaactgga caaagctgga ttcatcaaga ggcagctggt
     3001 ggaaacacgc cagatcacta aacacgtcgc acagattctg gatagtcgca tgaacacaaa
     3061 gtacgatgag aatgacaaac tgatcagaga agtgaaggtc attaccctga agagtaaact
     3121 ggtcagcgac tttaggaaag atttccagtt ttataaggtc cgcgagatta acaattatca
     3181 ccatgcccat gacgcatacc tgaacgccgt ggtcggtacc gcactgatca agaaataccc
     3241 aaaactggag agcgaattcg tgtacggaga ctataaggtg tacgatgtca gaaaaatgat
     3301 cgccaagtcc gagcaggaaa ttggaaaagc tactgccaag tatttctttt actcaaacat
     3361 catgaatttc tttaagacag agatcaccct ggccaatgga gaaatccgca aaaggcccct
     3421 gattgagaca aacggagaga caggcgaaat cgtgtgggac aaaggcagag attttgcaac
     3481 cgtgaggaag gtcctgagca tgcctcaagt gaatatcgtc aagaaaactg aggtgcagac
     3541 aggcggtttc tcaaaagaaa gtattctgcc aaaacgcaac tctgataagc tgatcgctag
     3601 aaagaaagac tgggacccta agaagtatgg aggctttgac tctcccactg tggcatactc
     3661 cgtcctggtg gtcgctaagg tggagaaggg caaaagcaag aaactgaaat ctgtcaagga
     3721 gctgctgggt atcacaatta tggagagaag ctctttcgag aagaacccaa tcgattttct
     3781 ggaggccaaa ggttataagg aagtgaagaa agacctgatc attaaactgc ccaagtacag
     3841 tctgtttgag ctggaaaacg gcaggaaacg catgctggca agcgctggag agctgcagaa
     3901 aggcaatgaa ctggccctgc cttctaagta cgtgaacttc ctgtatctgg caagccacta
     3961 cgagaagctg aaaggatctc cagaggataa cgaacagaaa cagctgtttg tggagcagca
     4021 caagcattat ctggacgaga tcattgaaca gattagcgag ttctctaaaa gagtgatcct
     4081 ggccgacgca aatctggata aggtcctgtc tgcttacaac aaacacagag ataagcccat
     4141 cagggagcag gccgaaaata tcattcatct gttcactctg acaaacctgg gcgcacctgc
     4201 agctttcaag tacttcgaca ctacaatcga tagaaagagg tacacctcca ctaaggaggt
     4261 gctggacgct acactgatcc atcagagtat taccggcctg tacgaaacaa ggattgacct
     4321 gtctcagctg ggtggcgact caccggtgag atctcctaag aagaagagaa aggtgggctc
     4381 tggggctaca aacttctctc tgctgaaaca agctggcgac gtggaggaga accctggacc
     4441 tgtgagcaag ggcgaggagg ataacatggc ctctctccca gcgacacatg agttacacat
     4501 ctttggctcc atcaacggtg tggactttga catggtgggt cagggcaccg gcaatccaaa
     4561 tgatggttat gaggagttaa acctgaagtc caccaagggt gacctccagt tctccccctg
     4621 gattctggtc cctcatatcg ggtatggctt ccatcagtac ctgccctacc ctgacgggat
     4681 gtcgcctttc caggccgcca tggtagatgg ctccggctac caagtccatc gcacaatgca
     4741 gtttgaagat ggtgcctccc ttactgttaa ctaccgctac acctacgagg gaagccacat
     4801 caaaggagag gcccaggtga aggggactgg tttccctgct gacggtcctg tgatgaccaa
     4861 ctcgctgacc gctgcggact ggtgcaggtc gaagaagact taccccaacg acaaaaccat
     4921 catcagtacc tttaagtgga gttacaccac tggaaatggc aagcgctacc ggagcactgc
     4981 gcggaccacc tacacctttg ccaagccaat ggcggctaac tatctgaaga accagccgat
     5041 gtacgtgttc cgtaagacgg agctcaagca ctccaagacc gagctcaact tcaaggagtg
     5101 gcaaaaggcc tttaccgatg tgatgggcat ggacgagctg tacaagtaag ctagaactat
     5161 agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc
     5221 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     5281 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     5341 tttcaggttc agggggaggt gtgggaggtt ttttaattcc tcaagagacc tctagaccag
     5401 ccaggacaga aatgcctcga cttcgctgct acccaaggtt gccgggtgac gcacaccgtg
     5461 gaaacggatg aaggcacgaa cccagtggac ataagcctgt tcggttcgta agctgtaatg
     5521 caagtagcgt atgcgctcac gcaactggtc cagaaccttg accgaacgca gcggtggtaa
     5581 cggcgcagtg gcggttttca tggcttgtta tgactgtttt tttggggtac agtctatgcc
     5641 tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag
     5701 caacgatgtt acgcagcagg gcagtcgccc taaaacaaag ttaaacatta tgagggaagc
     5761 ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg agcgccatct
     5821 cgaaccgacg ttgctggccg tacatttgta cggctccgca gtggatggcg gcctgaagcc
     5881 acacagtgat attgatttgc tggttacggt gaccgtaagg cttgatgaaa caacgcggcg
     5941 agctttgatc aacgaccttt tggaaacttc ggcttcccct ggagagagcg agattctccg
     6001 cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa
     6061 gcgcgaactg caatttggag aatggcagcg caatgacatt cttgcaggta tcttcgagcc
     6121 agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac atagcgttgc
     6181 cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg atctatttga
     6241 ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg gcgatgagcg
     6301 aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc
     6361 gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc agcccgtcat
     6421 acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct cgcgcgcaga
     6481 tcagttggaa gaatttgtcc actacgtgaa aggcgagatc accaaggtag tcggcaaata
     6541 accctcgagc cacccatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc
     6601 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6661 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6721 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6781 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6841 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6901 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6961 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     7021 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7081 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7141 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7201 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7261 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     7321 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     7381 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     7441 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     7501 caacgcaatt aatacgcgta ccgctagcca ggaagagttt gtagaaacgc aaaaaggcca
     7561 tccgtcagga tggccttctg cttagtttga tgcctggcag tttatggcgg gcgtcctgcc
     7621 cgccaccctc cgggccgttg cttcacaacg ttcaaatccg ctcccggcgg
//
