LOCUS       sequence-435941-        5007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a protein of interest fused to the C-terminus of 
            Flag-APEX3 for proximity labeling or TEM (unpublished).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5007)
  TITLE     Mammalian expression empty backbones
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5007)
  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: 
            "Unpublished"
COMMENT     Sequence Label: Flag-APEX3-C Cloning Vector
FEATURES             Location/Qualifiers
     source          1..5007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(251..1042,1043..1111))
                     /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     874..893
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1112..1216)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1242..1697)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1379..1400)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1591..1610
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1768..2752
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        2787..2805
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2787..2804
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2843..2866
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2873..3619
                     /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
                     YAADEDAFFADYAEAHQKASELGFADA"
     polyA_signal    3684..3818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3708..3727)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3762..3781
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3931..3951)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3931..3949)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3970..3986)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3970..3986)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3983..4005)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3994..4010
                     /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(4018..4024,4025..4042,4043..4048))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4063..4084
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4201..4218)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4372..4960)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4452..4471)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
       61 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
      121 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
      181 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
      241 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
      301 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
      361 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
      421 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
      481 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
      541 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
      601 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
      661 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
      721 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
      781 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
      841 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
      901 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
      961 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     1021 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     1081 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     1141 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     1201 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     1261 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     1321 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     1381 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     1441 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     1501 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     1561 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     1621 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     1681 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
     1741 ggtgcgggcc tcttcgctat tacgccagtc gaccgccaat tcaatatggc gtatatggac
     1801 tcatgccaat tcaatatggt ggatctggac ctgtgccaat tcaatatggc gtatatggac
     1861 tcgtgccaat tcaatatggt ggatctggac cccagccaat tcaatatggc ggacttggca
     1921 ccatgccaat tcaatatggc ggacctggca ctgtgccaac tggggagggg tctacttggc
     1981 acggtgccaa gtttgaggag gggtcttggc cctgtgccaa gtccgccata ttgaattggc
     2041 atggtgccaa taatggcggc catattggct atatgccagg atcaatatat aggcaatatc
     2101 caatatggcc ctatgccaat atggctattg gccaggttca atactatgta ttggccctat
     2161 gccatatagt attccatata tgggttttcc tattgacgta gatagcccct cccaatgggc
     2221 ggtcccatat accatatatg gggcttccta ataccgccca tagccactcc cccattgacg
     2281 tcaatggtct ctatatatgg tctttcctat tgacgtcata tgggcggtcc tattgacgta
     2341 tatggcgcct cccccattga cgtcaattac ggtaaatggc ccgcctggct caatgcccat
     2401 tgacgtcaat aggaccaccc accattgacg tcaatgggat ggctcattgc ccattcatat
     2461 ccgttctcac gccccctatt gacgtcaatg acggtaaatg gcccacttgg cagtacatca
     2521 atatctatta atagtaactt ggcaagtaca ttactattgg aagtacgcca gggtacattg
     2581 gcagtactcc cattgacgtc aatggcggta aatggcccgc gatggctgcc aagtacatcc
     2641 ccattgacgt caatggggag gggcaatgac gcaaatgggc gttccattga cgtaaatggg
     2701 cggtaggcgt gcctaatggg aggtctatat aagcaatgct cgtttaggga accgccattc
     2761 tgcctgggga cgtcggagca agcttgattt aggtgacact atagaataca agctacttgt
     2821 tctttttgca ggatccacca tggattacaa ggatgacgac gataagatcc gcggaaagtc
     2881 ttacccaact gtgagtgctg attaccagga cgccgttgag aaggcgaaga agaagctcag
     2941 aggcttcatc gctgagaaga gatgcgctcc tctaatgctc cgtttggcat tccactctgc
     3001 tggaaccttt gacaagggca cgaagactgg tggacccttc ggaaccatca agcaccctgc
     3061 cgaactggct cacagcgcta acaacggtct tgacatcgct gttaggcttt tggagccact
     3121 caaggcggag ttccctattt tgagctacgc cgatttctac cagttggctg gcgttgttgc
     3181 cgttgaggtc acgggtggac ctaaggttcc attccaccct ggaagagagg acaagcctga
     3241 gccaccacca gagggtcgct tgcctgatcc cactaagggt tctgaccatt tgagagatgt
     3301 gtttggcaaa gctatggggc ttactgacca agatattgtt gctctatctg ggggtcacac
     3361 tattggagct gcacacaagg agcgttctgg atttgagggt ccctggacct ctaatcctct
     3421 tattttcgac aactcatact tcacggagtt gttgagtggt gagaaggaag gtctccttca
     3481 gctaccttct gacaaggctc ttttgtctga ccctgtattc cgccctctcg ttgataaata
     3541 tgcagcggac gaagatgcct tctttgctga ttacgctgag gctcaccaaa aggcttccga
     3601 gcttgggttt gctgatgccg gatctcagat ctcgaattca aggcctctcg agcctctaga
     3661 actatagtga gtcgtattac gtagatccag acatgataag atacattgat gagtttggac
     3721 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     3781 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     3841 ttatgtttca ggttcagggg gaggtgtggg aggtttttta attcgcggcc gcggcgccaa
     3901 tgcattgggc ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     3961 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4021 catacgagcc gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4081 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4141 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4201 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4261 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4321 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4381 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4441 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4501 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4561 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4621 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4681 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4741 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4801 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4861 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4921 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4981 tacggggtct gacgctcagt ggaacga
//
