LOCUS       sequence-259784-        6245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Foundation cassette containing lox sites for cre-mediated exchange 
            of inducible vectors, confers mammalian G418 resistance, targets 
            AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6245)
  AUTHORS   Wang X, Friesen E, Muller I, Lemieux M, Dukart R, Maia IB, Kalia S, 
            Schmitt-Ulms G
  TITLE     Rapid Generation of Human Neuronal Cell Models Enabling Inducible 
            Expression of Proteins-of-interest for Functional Studies.
  JOURNAL   Bio Protoc. 2020 May 5;10(9):e3615. doi: 10.21769/BioProtoc.3615. 
            eCollection 2020 May 5.
  PUBMED    33659578
REFERENCE   2  (bases 1 to 6245)
  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: "Bio 
            Protoc."; date: "2020-05-5"; volume: "10(9):e3615. doi"; pages: " 
            10.21769/BioProtoc.3615. eCollection 2020 May 5"
COMMENT     Sequence Label: AAVS1 KanR foundation lox casette
FEATURES             Location/Qualifiers
     source          1..6245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      23..611
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     512..531
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     765..782
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    899..920
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(935..940,941..958,959..965)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    973..989
                     /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)."
     primer_bind     978..1000
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     997..1013
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     997..1013
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1034..1052
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    1087..1890
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    complement(1891..1924)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1927..1960
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    1965..1990
                     /label=SA
                     /note="splice acceptor site"
     CDS             2014..2067
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2086..2880
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWAYRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2140..2159)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2750..2769
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    2884..2917
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(3102..3121)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3146..3193
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    complement(3301..3334)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     misc_feature    3335..4171
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(4205..4224)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4206..4224)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4231..4248)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4231..4247)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4240..4262)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             4385..4687
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4566..4585
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(join(5284..6075,6076..6144))
                     /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     5907..5926
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
       61 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
      121 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
      181 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
      241 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
      301 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
      361 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
      421 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
      481 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
      541 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      601 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      661 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
      721 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
      781 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
      841 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
      901 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
      961 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     1021 acgccaagct cagaattaac cctcactaaa gggactagtc ctgcaggttt aaacgaattc
     1081 gccctttgct ttctctgacc agcattctct cccctgggcc tgtgccgctt tctgtctgca
     1141 gcttgtggcc tgggtcacct ctacggctgg cccagatcct tccctgccgc ctccttcagg
     1201 ttccgtcttc ctccactccc tcttcccctt gctctctgct gtgttgctgc ccaaggatgc
     1261 tctttccgga gcacttcctt ctcggcgctg caccacgtga tgtcctctga gcggatcctc
     1321 cccgtgtctg ggtcctctcc gggcatctct cctccctcac ccaaccccat gccgtcttca
     1381 ctcgctgggt tcccttttcc ttctccttct ggggcctgtg ccatctctcg tttcttagga
     1441 tggccttctc cgacggatgt ctcccttgcg tcccgcctcc ccttcttgta ggcctgcatc
     1501 atcaccgttt ttctggacaa ccccaaagta ccccgtctcc ctggctttag ccacctctcc
     1561 atcctcttgc tttctttgcc tggacacccc gttctcctgt ggattcgggt cacctctcac
     1621 tcctttcatt tgggcagctc ccctaccccc cttacctctc tagtctgtgc tagctcttcc
     1681 agccccctgt catggcatct tccaggggtc cgagagctca gctagtcttc ttcctccaac
     1741 ccgggcccct atgtccactt caggacagca tgtttgctgc ctccagggat cctgtgtccc
     1801 cgagctggga ccaccttata ttcccagggt cggttaatgt ggctctggtt ctgggtactt
     1861 ttatctgtcc cctccacccc acagtggggc taccgttcgt atagcataca ttatacgaag
     1921 ttataagaag ttcctattct ctagaaagta taggaacttc gcttctgacc tcttctcttc
     1981 ctcccacagg gcctcgagag atctggcagc ggagagggca gaggaagtct tctaacatgc
     2041 ggtgacgtgg aggagaatcc cggccctagg ctcgaggcga tcgcaatgat tgaacaagat
     2101 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     2161 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     2221 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     2281 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     2341 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     2401 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     2461 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     2521 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     2581 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     2641 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     2701 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     2761 cgtgatattg ctgaagagct tggcggcgaa tgggcttacc gcttcctcgt gctttacggt
     2821 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     2881 gcggaagttc ctattctcta gaaagtatag gaacttcgga ctctggggtt cgaaatgacc
     2941 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     3001 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     3061 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
     3121 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
     3181 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     3241 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacctcagc
     3301 ataacttcgt ataggatact ttatacgaag ttatactagg gacaggattg ttgacagaaa
     3361 agccccatcc ttaggcctcc tccttcctag tctcctgata ttgggtctaa cccccacctc
     3421 ctgttaggca gattccttat ctggtgacac acccccattt cctggagcca tctctctcct
     3481 tgccagaacc tctaaggttt gcttacgatg gagccagaga ggatcctggg agggagagct
     3541 tggcaggggg tgggagggaa gggggggatg cgtgacctgc ccggttctca gtggccaccc
     3601 tgcgctaccc tctcccagaa cctgagctgc tctgacgcgg ctgtctggtg cgtttcactg
     3661 atcctggtgc tgcagcttcc ttacacttcc caagaggaga agcagtttgg aaaaacaaaa
     3721 tcagaataag ttggtcctga gttctaactt tggctcttca cctttctagt ccccaattta
     3781 tattgttcct ccgtgcgtca gttttacctg tgagataagg ccagtagcca gccccgtcct
     3841 ggcagggctg tggtgaggag gggggtgtcc gtgtggaaaa ctccctttgt gagaatggtg
     3901 cgtcctaggt gttcaccagg tcgtggccgc ctctactccc tttctctttc tccatccttc
     3961 tttccttaaa gagtccccag tgctatctgg gacatattcc tccgcccaga gcagggtccc
     4021 gcttccctaa ggccctgctc tgggcttctg ggtttgagtc cttggcaagc ccaggagagg
     4081 cgctcaggct tccctgtccc ccttcctcgt ccaccatctc atgcccctgg ctctcctgcc
     4141 ccttccctac aggggttcct ggctctgctc taagggcgaa ttcgcggccg ctaaattcaa
     4201 ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg
     4261 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     4321 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctatacgt
     4381 acggcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt
     4441 acagagtgat attattgaca cgccggggcg acggatggtg atccccctgg ccagtgcacg
     4501 tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag
     4561 ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt
     4621 ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg
     4681 aatataaatg tcaggcatga gattatcaaa aaggatcttc acctagatcc ttttcacgta
     4741 gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg
     4801 gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg
     4861 atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc
     4921 ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat
     4981 ctgatggcgc aggggatcaa gctctgatca agagacagga tgaggatcgt ttcgattatt
     5041 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     5101 cgcatcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5161 aatacattca aatatgtatc cgctcatgag attatcaaaa aggatcttca cctagatcct
     5221 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5281 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5341 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5401 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5461 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5521 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5581 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5641 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5701 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5761 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5821 ttctgtgact ggtgaatact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5881 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5941 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6001 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6061 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6121 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6181 gggttattgt ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6241 ccccg
//
