LOCUS       sequence-400026-        7647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gamma-retroviral vector that expresses mNeonGreen fused to APEX2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7647)
  AUTHORS   Becker JT, Auerbach AA, Harris RS
  TITLE     APEX3 - an optimized tool for rapid and unbiased proximity labeling.
  JOURNAL   J Mol Biol. 2023 May 12:168145. doi: 10.1016/j.jmb.2023.168145.
  PUBMED    37182813
REFERENCE   2  (bases 1 to 7647)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Mol Biol.
            2023 May 12:168145. doi: 10.1016/j.jmb.2023.168145."
COMMENT     Sequence Label: mNeonGreen-APEX2
FEATURES             Location/Qualifiers
     source          1..7647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    580..921
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             988..1404
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1334..1356
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1372..1388
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1419..1428
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1425..2132
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     CDS             2187..2933
                     /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             2949..2990
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3191..3208)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     complement(4755..4771)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4755..4771)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4768..4790)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4779..4795
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4803..4809,4810..4827,4828..4833))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4848..4869
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4986..5003)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5157..5745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5237..5256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5916..6707,6708..6776))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6539..6558
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6777..6881)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6949..6967
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7005..7027)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7127..7146
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7340..7362
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7354..7371
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7355..7371
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7570..7589
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt
      301 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc
      361 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat
      421 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc
      481 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag
      541 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc
      601 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg
      661 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttctga
      721 acacccggcc gcaaccctgg gagacgtccc agggactttg ggggccgttt ttgtggcccg
      781 acctgaggaa gggagtcgat gtggaatccg accccgtcag gatatgtggt tctggtagga
      841 gacgagaacc taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttggaaccga
      901 agccgcgcgt cttgtctgct gcagcgctgc agcatcgttc tgtgttgtct ctgtctgact
      961 gtgtttctgt atttgtctga aaattagggc cagactgtta ccactccctt aagtttgacc
     1021 ttaggtcact ggaaagatgt cgagcggatc gctcacaacc agtcggtaga tgtcaagaag
     1081 agacgttggg ttaccttctg ctctgcagaa tggccaacct ttaacgtcgg atggccgcga
     1141 gacggcacct ttaaccgaga cctcatcacc caggttaaga tcaaggtctt ttcacctggc
     1201 ccgcatggac acccagacca ggtcccctac atcgtgacct gggaagcctt ggcttttgac
     1261 ccccctccct gggtcaagcc ctttgtacac cctaagcctc cgcctcctct tcctccatcc
     1321 gccccgtctc tcccccttga acctcctcgt tcgaccccgc ctcgatcctc cctttatcca
     1381 gccctcactc cttctctagg cgcctgtatg aattcgccgc caccatggtg agcaagggcg
     1441 aggaggataa catggcctct ctcccagcga cacatgagtt acacatcttt ggctccatca
     1501 acggtgtgga ctttgacatg gtgggtcagg gcaccggcaa tcccaatgat ggttatgagg
     1561 agttaaacct gaagtccacc aagggtgacc tccagttctc cccctggatt ctggtccctc
     1621 atatcgggta tggcttccat cagtacctgc cctaccctga cgggatgtcg cctttccagg
     1681 ccgcgatggt agatggctcc ggataccaag tccatcgcac aatgcagttt gaagatggtg
     1741 cctcccttac tgtaaactac cgctacacct acgagggaag ccacatcaaa ggagaggccc
     1801 aggtgaaggg gactggtttc cctgctgacg gtcctgtgat gaccaactcg ctgaccgctg
     1861 cggactggtg caggtcgaag aagacttacc ccaacgacaa aaccatcatc agtaccttta
     1921 agtggagtta caccactgga aatggcaagc gctaccggag cactgcgcgg accacctaca
     1981 cctttgccaa gccaatggcg gctaactatc tgaagaacca gccgatgtac gtgttccgta
     2041 agacggagct gaagcactcc aagaccgagc tgaacttcaa ggagtggcaa aaggccttta
     2101 ccgatgtgat gggcatggac gagctgtaca agggtggcgg tgggtcaggt ggcggcggaa
     2161 cgcgtaccgg tggaggcggg ggttctggaa agtcttaccc aactgtgagt gctgattacc
     2221 aggacgccgt tgagaaggcg aagaagaagc tcagaggctt catcgctgag aagagatgcg
     2281 ctcctctaat gctccgtttg gcattccact ctgctggaac ctttgacaag ggcacgaaga
     2341 ctggtggacc cttcggaacc atcaagcacc ctgccgaact ggctcacagc gctaacaacg
     2401 gtcttgacat cgctgttagg cttttggagc cactcaaggc ggagttccct attttgagct
     2461 acgccgattt ctaccagttg gctggcgttg ttgccgttga ggtcacgggt ggacctaagg
     2521 ttccattcca ccctggaaga gaggacaagc ctgagccacc accagagggt cgcttgcctg
     2581 atcccactaa gggttctgac catttgagag atgtgtttgg caaagctatg gggcttactg
     2641 accaagatat tgttgctcta tctgggggtc acactattgg agctgcacac aaggagcgtt
     2701 ctggatttga gggtccctgg acctctaatc ctcttatttt cgacaactca tacttcacgg
     2761 agttgttgag tggtgagaag gaaggtctcc ttcagctacc ttctgacaag gctcttttgt
     2821 ctgaccctgt attccgccct ctcgttgata aatatgcagc ggacgaagat gccttctttg
     2881 ctgattacgc tgaggctcac caaaagctat ccgagcttgg gtttgctgat gccggagggg
     2941 gtggctctgg taagcctatc cctaaccctc tcctcggtct cgattctacg taactcgagt
     3001 taatgttaac taactgacta gcgcccctct ccctcccccc cccctaacgt tactggccga
     3061 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg
     3121 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg
     3181 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt
     3241 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac
     3301 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca
     3361 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg
     3421 ctctcctcaa gcgtagtcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg
     3481 ggaatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa
     3541 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atggccaagc
     3601 cactgtccca agaagagtca actctgatcg agagggccac tgcaaccatt aatagcattc
     3661 ccatctctga agactatagc gtagctagtg ccgcactcag ctctgatgga cgcatattca
     3721 ccggcgttaa tgtctaccac ttcaccggcg gaccctgcgc cgaactggtc gtgctgggga
     3781 ccgcagccgc cgcggctgcc gggaatttga cgtgcattgt tgcaataggc aacgagaata
     3841 ggggcatcct gtcaccttgc ggccggtgtc ggcaagtgct gctggacctg caccccggca
     3901 tcaaggccat agtcaaggat agtgatggcc agccgaccgc cgttgggatt cgagaacttc
     3961 tgccttctgg gtacgtctgg gaaggctagg ctaagctcgc tcggctgggt cgcgggaggg
     4021 ccggtcgcag tgattcaggc ccttctggat tgtgttggtc cccagggcac gattgtcatg
     4081 cccacgcact cgggtgatct gactgatccc gcagattgga gatcgccgcc cgtgcctgcc
     4141 gattgggtgc agatccatgt cgacctgcag ccaagcttat cgataaaata aaagatttta
     4201 tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct
     4261 taagtaacgc cattttgcaa ggcatggaaa atacataact gagaatagag aagttcagat
     4321 caaggttagg aacagagaga cagcagaata tgggccaaac aggatatctg tggtaagcag
     4381 ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca
     4441 gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaaa tgaccctgtg
     4501 ccttatttga actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc
     4561 gagctcaata aaagagccca caacccctca ctcggcgcgc cagtcctccg atagactgcg
     4621 tcgcccgggt acccgtgtat ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc
     4681 gctgttcctt gggagggtct cctctgagtg attgactacc cgtcagcggg ggtctttcag
     4741 aattggtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4801 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4861 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4921 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4981 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5041 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5101 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5161 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5221 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5281 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5341 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5401 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5461 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5521 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5581 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5641 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5701 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5761 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5821 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5881 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5941 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6001 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6061 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6121 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6181 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6241 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6301 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6361 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6421 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6481 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6541 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6601 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6661 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6721 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6781 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6841 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6901 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     6961 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     7021 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     7081 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     7141 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     7201 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     7261 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     7321 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgccacgctc
     7381 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     7441 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     7501 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     7561 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     7621 gccggccacg atgcgtccgg cgtagag
//
