LOCUS       sequence-224265-        7225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Creation of a stable cell line expressing APP mutation (AD in vitro 
            study).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7225)
  TITLE     Radenovic Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7225)
  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            "Unpublished"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: pc DNA FRT TO- APP695
FEATURES             Location/Qualifiers
     source          1..7225
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        161..540
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        541..744
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     695..715
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    746..764
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    767..785
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     795..819
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             complement(1281..1292)
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="NHKV"
     CDS             1772..1792
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     primer_bind     complement(3103..3120)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3109..3333
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3466..3485)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3617..3664
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3672..4694
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3672..4694
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4824..4945
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4861..4880)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4915..4934
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4994..5010)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4994..5010)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5007..5029)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5018..5034
                     /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(5042..5048,5049..5066,5067..5072))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5087..5108
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5225..5242)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5396..5984)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5476..5495)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6155..6946,6947..7015))
                     /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     6778..6797
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7016..7120)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7195..7214)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
       61 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
      121 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
      181 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      241 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      301 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      361 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      421 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      481 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      541 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      601 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      661 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      721 tgggaggtct atataagcag agctctccct atcagtgata gagatctccc tatcagtgat
      781 agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
      841 tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa
      901 cttaagcttg gtaccgagct cggatccact agtccagtgt ggtggaattc tgcatgctgc
      961 ccggtttggc actgctcctg ctggccgcct ggacggctcg ggcgctggag gtacccactg
     1021 atggtaatgc tggcctgctg gctgaacccc agattgccat gttctgtggc agactgaaca
     1081 tgcacatgaa tgtccagaat gggaagtggg attcagatcc atcagggacc aaaacctgca
     1141 ttgataccaa ggaaggcatc ctgcagtatt gccaagaagt ctaccctgaa ctgcagatca
     1201 ccaatgtggt agaagccaac caaccagtga ccatccagaa ctggtgcaag cggggccgca
     1261 agcagtgcaa gacccatccc cactttgtga ttccctaccg ctgcttagtt ggtgagtttg
     1321 taagtgatgc ccttctcgtt cctgacaagt gcaaattctt acaccaggag aggatggatg
     1381 tttgcgaaac tcatcttcac tggcacaccg tcgccaaaga gacatgcagt gagaagagta
     1441 ccaacttgca tgactacggc atgttgctgc cctgcggaat tgacaagttc cgaggggtag
     1501 agtttgtgtg ttgcccactg gctgaagaaa gtgacaatgt ggattctgct gatgcggagg
     1561 aggatgactc ggatgtctgg tggggcggag cagacacaga ctatgcagat gggagtgaag
     1621 acaaagtagt agaagtagca gaggaggaag aagtggctga ggtggaagaa gaagaagccg
     1681 atgatgacga ggacgatgag gatggtgatg aggtagagga agaggctgag gaaccctacg
     1741 aagaagccac agagagaacc accagcattg ccaccaccac caccaccacc acagagtctg
     1801 tggaagaggt ggttcgagtt cctacaacag cagccagtac ccctgatgcc gttgacaagt
     1861 atctcgagac acctggggat gagaatgaac atgcccattt ccagaaagcc aaagagaggc
     1921 ttgaggccaa gcaccgagag agaatgtccc aggtcatgag agaatgggaa gaggcagaac
     1981 gtcaagcaaa gaacttgcct aaagctgata agaaggcagt tatccagcat ttccaggaga
     2041 aagtggaatc tttggaacag gaagcagcca acgagagaca gcagctggtg gagacacaca
     2101 tggccagagt ggaagccatg ctcaatgacc gccgccgcct ggccctggag aactacatca
     2161 ccgctctgca ggctgttcct cctcggcctc gtcacgtgtt caatatgcta aagaagtatg
     2221 tccgcgcaga acagaaggac agacagcaca ccctaaagca tttcgagcat gtgcgcatgg
     2281 tggatcccaa gaaagccgct cagatccggt cccaggttat gacacacctc cgtgtgattt
     2341 atgagcgcat gaatcagtct ctctccctgc tctacaacgt gcctgcagtg gccgaggaga
     2401 ttcaggatga agttgatgag ctgcttcaga aagagcaaaa ctattcagat gacgtcttgg
     2461 ccaacatgat tagtgaacca aggatcagtt acggaaacga tgctctcatg ccatctttga
     2521 ccgaaacgaa aaccaccgtg gagctccttc ccgtgaatgg agagttcagc ctggacgatc
     2581 tccagccgtg gcattctttt ggggctgact ctgtgccagc caacacagaa aacgaagttg
     2641 agcctgttga tgcccgccct gctgccgacc gaggactgac cactcgacca ggttctgggt
     2701 tgacaaatat caagacggag gagatctctg aagtgaagat ggatgcagaa ttccgacatg
     2761 actcaggata tgaagttcat catcaaaaat tggtgttctt tgcagaagat gtgggttcaa
     2821 acaaaggtgc aatcattgga ctcatggtgg gcggtgttgt catagcgaca gtgatcgtca
     2881 tcaccttggt gatgctgaag aagaaacagt acacatccat tcatcatggt gtggtggagg
     2941 ttgacgccgc tgtcacccca gaggagcgcc acctgtccaa gatgcagcag aacggctacg
     3001 aaaatccaac ctacaagttc tttgagcaga tgcagaacta gagatatcca gcacattggc
     3061 ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     3121 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3181 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3241 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3301 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     3361 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3421 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3481 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3541 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3601 atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa agtataggaa
     3661 cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa
     3721 agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca
     3781 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct
     3841 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc
     3901 ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
     3961 tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg
     4021 ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac
     4081 cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc
     4141 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc
     4201 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
     4261 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
     4321 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt
     4381 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag
     4441 gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct
     4501 tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
     4561 gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga
     4621 ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga
     4681 gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     4741 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4801 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4861 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4921 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4981 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5041 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5101 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5161 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5221 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5281 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5341 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5401 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5461 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5521 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5581 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5641 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5701 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5761 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5821 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5881 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5941 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6001 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6061 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6121 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6181 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6241 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6301 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6361 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6421 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6481 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6541 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6601 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6661 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6721 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6781 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6841 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6901 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6961 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7021 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7081 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7141 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     7201 ctgctctgat gccgcatagt taagc
//
