LOCUS       sequence-57673-e        6198 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6198)
  AUTHORS   Rhee HW, Zou P, Udeshi ND, Martell JD, Mootha VK, Carr SA, Ting AY
  TITLE     Proteomic Mapping of Mitochondria in Living Cells via Spatially 
            Restricted Enzymatic Tagging.
  JOURNAL   Science. 2013 Jan 31.
  PUBMED    23371551
REFERENCE   2  (bases 1 to 6198)
  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: "Science. 
            2013 Jan 31."
COMMENT     Sequence Label: pcDNA3-APX(W41F)-NES
FEATURES             Location/Qualifiers
     source          1..6198
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             910..933
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             934..1680
                     /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="GKSYPTVSADYQKAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPEVPFHPGREDKPEPPPEGRLPDATKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     CDS             1687..1713
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(1750..1768)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1751..1768)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1788..1805)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1794..2018
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2064..2492
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2151..2170)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2361..2382
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2501..2521)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2506..2836
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2687..2822
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2749..2768
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2903..3697
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2957..2976)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3567..3586
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3871..3992
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3908..3927)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3962..3981
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4041..4057)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4041..4057)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4054..4076)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4065..4081
                     /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(4089..4095,4096..4113,4114..4119))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4134..4155
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4272..4289)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4443..5031)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4523..4542)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5202..5993,5994..6062))
                     /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     5825..5844
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6063..6167)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg actacaagga tgacgacgat aagggaaagt cttacccaac tgtgagtgct
      961 gattaccaga aggccgttga gaaggcgaag aagaagctca gaggcttcat cgctgagaag
     1021 agatgcgctc ctctaatgct ccgtttggca ttccactctg ctggaacctt tgacaagggc
     1081 acgaagaccg gtggaccctt cggaaccatc aagcaccctg ccgaactggc tcacagcgct
     1141 aacaacggtc ttgacatcgc tgttaggctt ttggagccac tcaaggcgga gttccctatt
     1201 ttgagctacg ccgatttcta ccagttggct ggcgttgttg ccgttgaggt cacgggtgga
     1261 cctgaagttc cattccaccc tggaagagag gacaagcctg agccaccacc agagggtcgc
     1321 ttgcccgatg ccactaaggg ttctgaccat ttgagagatg tgtttggcaa agctatgggg
     1381 cttactgacc aagatatcgt tgctctatct gggggtcaca ctattggagc tgcacacaag
     1441 gagcgttctg gatttgaggg tccctggacc tctaatcctc ttattttcga caactcatac
     1501 ttcacggagt tgttgagtgg tgagaaggaa ggtctccttc agctaccttc tgacaaggct
     1561 cttttgtctg accctgtatt ccgccctctc gttgataaat atgcagcgga cgaagatgcc
     1621 ttctttgctg attacgctga ggctcaccaa aagctttccg agcttgggtt tgctgatgcc
     1681 ctgcagctgc cccccctgga gcgcctgacc ctggactaat agctcgagca tgcatctaga
     1741 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     1801 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1861 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     1921 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     1981 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2041 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2101 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2161 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2221 catcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2281 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2341 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2401 ctcggtctat tcttttgatt tataagggat tttggggatt tcggcctatt ggttaaaaaa
     2461 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     2521 tgtggaaagt ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta
     2581 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2641 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     2701 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     2761 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     2821 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     2881 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     2941 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3001 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3061 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     3121 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     3181 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     3241 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     3301 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     3361 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     3421 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     3481 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     3541 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     3601 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     3661 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     3721 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     3781 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     3841 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     3901 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     3961 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4021 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4081 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4141 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4201 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4261 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4321 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4381 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4441 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4501 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4561 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4621 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4681 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4741 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4801 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4861 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     4921 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4981 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5041 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5101 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5161 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5221 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5281 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5341 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5401 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5461 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5521 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5581 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5641 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5701 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5761 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5821 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5881 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5941 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6001 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6061 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6121 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6181 aaagtgccac ctgacgtc
//
