LOCUS       sequence-135875-        8007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GCaMP6f knock in construct into the AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8007)
  AUTHORS   Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan
            AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen 
            TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP, 
            Weissman JS, Qi LS, So PL, Conklin BR
  TITLE     CRISPR Interference Efficiently Induces Specific and Reversible Gene
            Silencing in Human iPSCs.
  JOURNAL   Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi: 
            10.1016/j.stem.2016.01.022. Epub 2016 Mar 10.
  PUBMED    26971820
REFERENCE   2  (bases 1 to 8007)
  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"; doi: 
            "10.1016/j.stem.2016.01.022"; journalName: "Cell Stem Cell"; date: 
            "2016-04-7- 7"; volume: "18"; issue: "4"; pages: "541-53"
COMMENT     Sequence Label: pAAVS1-PC-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..8007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..804
                     /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(806..839)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    846..871
                     /label=SA
                     /note="splice acceptor site"
     CDS             895..948
                     /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             958..1557
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(958..977)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1454..1474
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1589..1606)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1595..1819
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        1825..2204
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2206..2483
                     /label=chicken beta-actin promoter
     intron          2485..3502
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3426..3448
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3510..3529
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3562..3571
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3583..3639
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     3852..3873
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3979..4000)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4240..4259)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(4908..4927)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4973..5028
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5027..5046)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    complement(5377..5410)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5451..6287
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     rep_origin      complement(6335..6923)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6415..6434)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7094..7885,7886..7954))
                     /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     7717..7736
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgctttctct gaccagcatt ctctcccctg ggcctgtgcc gctttctgtc tgcagcttgt
       61 ggcctgggtc acctctacgg ctggcccaga tccttccctg ccgcctcctt caggttccgt
      121 cttcctccac tccctcttcc ccttgctctc tgctgtgttg ctgcccaagg atgctctttc
      181 cggagcactt ccttctcggc gctgcaccac gtgatgtcct ctgagcggat cctccccgtg
      241 tctgggtcct ctccgggcat ctctcctccc tcacccaacc ccatgccgtc ttcactcgct
      301 gggttccctt ttccttctcc ttctggggcc tgtgccatct ctcgtttctt aggatggcct
      361 tctccgacgg atgtctccct tgcgtcccgc ctccccttct tgtaggcctg catcatcacc
      421 gtttttctgg acaaccccaa agtaccccgt ctccctggct ttagccacct ctccatcctc
      481 ttgctttctt tgcctggaca ccccgttctc ctgtggattc gggtcacctc tcactccttt
      541 catttgggca gctcccctac cccccttacc tctctagtct gtgctagctc ttccagcccc
      601 ctgtcatggc atcttccagg ggtccgagag ctcagctagt cttcttcctc caacccgggc
      661 ccctatgtcc acttcaggac agcatgtttg ctgcctccag ggatcctgtg tccccgagct
      721 gggaccacct tatattccca gggccggtta atgtggctct ggttctgggt acttttatct
      781 gtcccctcca ccccacagtg gggcaataac ttcgtatagc atacattata cgaagttata
      841 agcttctgac ctcttctctt cctcccacag ggcctcgaga gatctggcag cggagagggc
      901 agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag gctcgagatg
      961 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     1021 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     1081 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     1141 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     1201 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     1261 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     1321 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     1381 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     1441 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     1501 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgatct
     1561 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     1621 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1681 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1741 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     1801 gatgcggtgg gctctatggg tctcgacatt gattattgac tagttattaa tagtaatcaa
     1861 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     1921 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1981 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2041 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2101 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     2161 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
     2221 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
     2281 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg
     2341 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
     2401 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     2461 aaaagcgaag cgcgcggcgg gcggggagtc gctgcgacgc tgccttcgcc ccgtgccccg
     2521 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
     2581 gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct
     2641 tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg
     2701 ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc
     2761 cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag
     2821 tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg
     2881 gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc
     2941 ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc
     3001 gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg
     3061 gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg
     3121 gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct
     3181 tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc
     3241 gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc
     3301 cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct
     3361 ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag
     3421 ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca
     3481 tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca
     3541 ttttggcaaa gaatgtcgac cgccaccatg gttgactcat cacgtcgtaa gtggaataag
     3601 acaggtcacg cagtcagagc tataggtcgg ctgagctcac tcgagaacgt ctatatcaag
     3661 gccgacaagc agaagaacgg catcaaggcg aacttcaaga tccgccacaa catcgaggac
     3721 ggcggcgtgc agctcgccta ccactaccag cagaacaccc ccatcggcga cggccccgtg
     3781 ctgctgcccg acaaccacta cctgagcgtg cagtccaaac tttcgaaaga ccccaacgag
     3841 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     3901 gacgagctgt acaagggcgg taccggaggg agcatggtga gcaagggcga ggagctgttc
     3961 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     4021 gtgtccggcg agggtgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     4081 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     4141 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     4201 cccgaaggct acatccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     4261 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     4321 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacct gccggaccaa
     4381 ctgactgaag agcagatcgc agaatttaaa gaggaattct ccctatttga caaggacggg
     4441 gatgggacaa taacaaccaa ggagctgggg acggtgatgc ggtctctggg gcagaacccc
     4501 acagaagcag agctgcagga catgatcaat gaagtagatg ccgacggtga cggcacaatc
     4561 gacttccctg agttcctgac aatgatggca agaaaaatga aatacaggga cacggaagaa
     4621 gaaattagag aagcgttcgg tgtgtttgat aaggatggca atggctacat cagtgcagca
     4681 gagcttcgcc acgtgatgac aaaccttgga gagaagttaa cagatgaaga ggttgatgaa
     4741 atgatcaggg aagcagacat cgatggggat ggtcaggtaa actacgaaga gtttgtacaa
     4801 atgatgacag cgaagtgaac gcgtgaattc actcctcagg tgcaggctgc ctatcagaag
     4861 gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct
     4921 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     4981 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     5041 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     5101 tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg
     5161 aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt
     5221 ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat
     5281 gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt
     5341 ctcttatgga gatccctcga cctgcagccc aagcttataa cttcgtatag catacattat
     5401 acgaagttat gcggccgcac cggtatgcat ttaattaaga tatcagcttt actagggaca
     5461 ggattggtga cagaaaagcc ccatccttag gcctcctcct tcctagtctc ctgatattgg
     5521 gtctaacccc cacctcctgt taggcagatt ccttatctgg tgacacaccc ccatttcctg
     5581 gagccatctc tctccttgcc agaacctcta aggtttgctt acgatggagc cagagaggat
     5641 cctgggaggg agagcttggc agggggtggg agggaagggg gggatgcgtg acctgcccgg
     5701 ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg agctgctctg acgcggctgt
     5761 ctggtgcgtt tcactgatcc tggtgctgca gcttccttac acttcccaag aggagaagca
     5821 gtttggaaaa acaaaatcag aataagttgg tcctgagttc taactttggc tcttcacctt
     5881 tctagtcccc aatttatatt gttcctccgt gcgtcagttt tacctgtgag ataaggccag
     5941 tagccagccc cgtcctggca gggctgtggt gaggaggggg gtgtccgtgt ggaaaactcc
     6001 ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt ggccgcctct actccctttc
     6061 tctttctcca tccttctttc cttaaagagt ccccagtgct atctgggaca tattcctccg
     6121 cccagagcag ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt tgagtccttg
     6181 gcaagcccag gagaggcgct caggcttccc tgtccccctt cctcgtccac catctcatgc
     6241 ccctggctct cctgcccctt ccctacaggg gttcctggct ctgctctaag ggcgaattcg
     6301 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6361 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6421 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6481 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6541 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6601 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6661 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6721 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     6781 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6841 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6901 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6961 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     7021 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     7081 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     7141 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     7201 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     7261 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     7321 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7381 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7441 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7501 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7561 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7621 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7681 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7741 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7801 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7861 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7921 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7981 gcatttatca gggttgaatt cgccctt
//
