LOCUS       sequence-386447-        8031 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses APP695 with a biotin Acceptor Peptide tag at N-terminal 
            for single molecule tracking and other measusers in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8031)
  AUTHORS   Capitini C, Bigi A, Parenti N, Emanuele M, Bianchi N, Cascella R, 
            Cecchi C, Maggi L, Annunziato F, Pavone FS, Calamai M
  TITLE     APP and Bace1: Differential effect of cholesterol enrichment on 
            processing and plasma membrane mobility.
  JOURNAL   iScience. 2023 Apr 11;26(5):106611. doi: 10.1016/j.isci.2023.106611.
            eCollection 2023 May 19.
  PUBMED    37128606
REFERENCE   2  (bases 1 to 8031)
  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: "iScience.";
            date: "2023-04-11"; volume: "26(5):106611. doi"; pages: " 
            10.1016/j.isci.2023.106611. eCollection 2023 May 19"
COMMENT     Sequence Label: AP-APP
FEATURES             Location/Qualifiers
     source          1..8031
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     151..173
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     165..182
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     166..182
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        343..722
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        723..926
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     877..897
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     952..971
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        952..970
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1089..1133
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             complement(1422..1433)
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="NHKV"
     CDS             1913..1933
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             3201..3227
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3236..3252)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    3263..3885
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3462..3481)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3909..3929)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3914..4243
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4094..4229
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4310..5104
                     /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(4364..4383)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4974..4993
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5278..5399
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5315..5334)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5369..5388
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5448..5464)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5461..5483)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5472..5488
                     /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(5496..5502,5503..5520,5521..5526))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5541..5562
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5679..5696)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5850..6438)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5930..5949)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6609..7400,7401..7469))
                     /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     7232..7251
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7470..7574)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7601..8029
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7688..7707)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7898..7919
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag
       61 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
      121 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
      181 gtgccaagct gatctataca ttgaatcaat attggcaatt agccatatta gtcattggtt
      241 atatagcata aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata
      301 tgtacattta tattggctca tgtccaatat gaccgccatg ttgacattga ttattgacta
      361 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      421 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      481 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      541 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      601 gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      661 tgaccttacg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      721 tggtgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat
      781 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      841 actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac
      901 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagaatttt gtaatacgac
      961 tcactatagg gcggccggga attcgtcgac tggatccggt accgaggaga tctgccgccg
     1021 cgatcgccat gctgcccggt ttggcactgc tcctgctggc cgcctggacg gctcgggcgg
     1081 ctagcgaggg cctgaacgat atcttcgagg cccagaagat cgagtggcac gagagtggcg
     1141 cgcctctgga ggtacccact gatggtaatg ctggcctgct ggctgaaccc cagattgcca
     1201 tgttctgtgg cagactgaac atgcacatga atgtccagaa tgggaagtgg gattcagatc
     1261 catcagggac caaaacctgc attgatacca aggaaggcat cctgcagtat tgccaagaag
     1321 tctaccctga actgcagatc accaatgtgg tagaagccaa ccaaccagtg accatccaga
     1381 actggtgcaa gcggggccgc aagcagtgca agacccatcc ccactttgtg attccctacc
     1441 gctgcttagt tggtgagttt gtaagtgatg cccttctcgt tcctgacaag tgcaaattct
     1501 tacaccagga gaggatggat gtttgcgaaa ctcatcttca ctggcacacc gtcgccaaag
     1561 agacatgcag tgagaagagt accaacttgc atgactacgg catgttgctg ccctgcggaa
     1621 ttgacaagtt ccgaggggta gagtttgtgt gttgcccact ggctgaagaa agtgacaatg
     1681 tggattctgc tgatgcggag gaggatgact cggatgtctg gtggggcgga gcagacacag
     1741 actatgcaga tgggagtgaa gacaaagtag tagaagtagc agaggaggaa gaagtggctg
     1801 aggtggaaga agaagaagcc gatgatgacg aggacgatga ggatggtgat gaggtagagg
     1861 aagaggctga ggaaccctac gaagaagcca cagagagaac caccagcatt gccaccacca
     1921 ccaccaccac cacagagtct gtggaagagg tggttcgagt tcctacaaca gcagccagta
     1981 cccctgatgc cgttgacaag tatctcgaga cacctgggga tgagaatgaa catgcccatt
     2041 tccagaaagc caaagagagg cttgaggcca agcaccgaga gagaatgtcc caggtcatga
     2101 gagaatggga agaggcagaa cgtcaagcaa agaacttgcc taaagctgat aagaaggcag
     2161 ttatccagca tttccaggag aaagtggaat ctttggaaca ggaagcagcc aacgagagac
     2221 agcagctggt ggagacacac atggccagag tggaagccat gctcaatgac cgccgccgcc
     2281 tggccctgga gaactacatc accgctctgc aggctgttcc tcctcggcct cgtcacgtgt
     2341 tcaatatgct aaagaagtat gtccgcgcag aacagaagga cagacagcac accctaaagc
     2401 atttcgagca tgtgcgcatg gtggatccca agaaagccgc tcagatccgg tcccaggtta
     2461 tgacacacct ccgtgtgatt tatgagcgca tgaatcagtc tctctccctg ctctacaacg
     2521 tgcctgcagt ggccgaggag attcaggatg aagttgatga gctgcttcag aaagagcaaa
     2581 actattcaga tgacgtcttg gccaacatga ttagtgaacc aaggatcagt tacggaaacg
     2641 atgctctcat gccatctttg accgaaacga aaaccaccgt ggagctcctt cccgtgaatg
     2701 gagagttcag cctggacgat ctccagccgt ggcattcttt tggggctgac tctgtgccag
     2761 ccaacacaga aaacgaagtt gagcctgttg atgcccgccc tgctgccgac cgaggactga
     2821 ccactcgacc aggttctggg ttgacaaata tcaagacgga ggagatctct gaagtgaaga
     2881 tggatgcaga attccgacat gactcaggat atgaagttca tcatcaaaaa ttggtgttct
     2941 ttgcagaaga tgtgggttca aacaaaggtg caatcattgg actcatggtg ggcggtgttg
     3001 tcatagcgac agtgatcgtc atcaccttgg tgatgctgaa gaagaaacag tacacatcca
     3061 ttcatcatgg tgtggtggag gttgacgccg ctgtcacccc agaggagcgc cacctgtcca
     3121 agatgcagca gaacggctac gaaaatccaa cctacaagtt ctttgagcag atgcagaaca
     3181 cgcgtacgcg gccgctcgag tatccgtacg acgtaccaga ctacgcataa ccgcggtcat
     3241 agctgtttcc tgaacagatc ccgggtggca tccctgtgac ccctccccag tgcctctcct
     3301 ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat
     3361 cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag
     3421 caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg
     3481 gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct
     3541 cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt
     3601 tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta
     3661 atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac
     3721 tgctcccttc cctgtccttc tgattttaaa ataactatac cagcaggagg acgtccagac
     3781 acagcatagg ctacctggcc atgcccaacc ggtgggacat ttgagttgct tgcttggcac
     3841 tgtcctctca tgcgttgggt ccactcagta gatgcctgtt gaattgggta cgcggccagc
     3901 ttggctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3961 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     4021 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     4081 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4141 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     4201 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     4261 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4321 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4381 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4441 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     4501 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4561 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4621 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4681 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4741 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4801 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     4861 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4921 tggattcatc gactgtggcc ggctgggtgt ggccgaccgc tatcaggaca tagcgttggc
     4981 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     5041 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     5101 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     5161 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     5221 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     5281 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     5341 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     5401 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     5461 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5521 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5581 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5641 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5701 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5761 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5821 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5881 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5941 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6001 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6061 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6121 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6181 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6241 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6301 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6361 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6421 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6481 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6541 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6601 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6661 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6721 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6781 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6841 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6901 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6961 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7021 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7081 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7141 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7201 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7261 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7321 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7381 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7441 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7501 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7561 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg tagcggcgca
     7621 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     7681 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     7741 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     7801 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     7861 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     7921 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     7981 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt t
//
