LOCUS       sequence-202850-        5959 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Homology arms, promoter and linker-mTagRFPT-CAAX sequence for 
            internal insertion of CAGGS-driven mTagRFPT-CAAX at the AAVS1 safe 
            harbor location in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5959)
  AUTHORS   Allen Institute for Cell Science
  TITLE     Allen Institute for Cell Science Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5959)
  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            "Unpublished"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: AICSDP-42:AAVS1-mTagRFPT-CAAX
FEATURES             Location/Qualifiers
     source          1..5959
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     misc_feature    188..689
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    816..841
                     /label=SA
                     /note="splice acceptor site"
     CDS             865..918
                     /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             928..1527
                     /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(928..947)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1424..1444
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1559..1576)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1565..1789
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        1795..2174
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2176..2453
                     /label=chicken beta-actin promoter
     intron          2455..3472
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3396..3418
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3480..3499
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3528..3537
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3534..4265
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     primer_bind     complement(4454..4473)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4519..4574
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4573..4592)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4935..4951)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4935..4951)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4948..4970)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4959..4975
                     /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(4983..4989,4990..5007,5008..5013))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5028..5049
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    5123..5959
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
ORIGIN
        1 ccctttgctt tctctgacca gcattctctc ccctgggcct gtgccgcttt ctgtctgcag
       61 cttgtggcct gggtcacctc tacggctggc ccagatcctt ccctgccgcc tccttcaggt
      121 tccgtcttcc tccactccct cttccccttg ctctctgctg tgttgctgcc caaggatgct
      181 ctttccggag cacttccttc tcggcgctgc accacgtgat gtcctctgag cggatcctcc
      241 ccgtgtctgg gtcctctccg ggcatctctc ctccctcacc caaccccatg ccgtcttcac
      301 tcgctgggtt cccttttcct tctccttctg gggcctgtgc catctctcgt ttcttaggat
      361 ggccttctcc gacggatgtc tcccttgcgt cccgcctccc cttcttgtag gcctgcatca
      421 tcaccgtttt tctggacaac cccaaagtac cccgtctccc tggctttagc cacctctcca
      481 tcctcttgct ttctttgcct ggacaccccg ttctcctgtg gattcgggtc acctctcact
      541 cctttcattt gggcagctcc cctacccccc ttacctctct agtctgtgct agctcttcca
      601 gccccctgtc atggcatctt ccaggggtcc gagagctcag ctagtcttct tcctccaacc
      661 cgggccccta tgtccacttc aggacagcat gtttgctgcc tccagggatc ctgtgtcccc
      721 gagctgggac caccttatat tcccagggcc ggttaatgtg gctctggttc tgggtacttt
      781 tatctgtccc ctccacccca cagtgggcca agcttctgac ctcttctctt cctcccacag
      841 ggcctcgaga gatctggcag cggagagggc agaggaagtc ttctaacatg cggtgacgtg
      901 gaggagaatc ccggccctag gctcgagatg accgagtaca agcccacggt gcgcctcgcc
      961 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     1021 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     1081 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     1141 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     1201 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     1261 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     1321 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     1381 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     1441 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     1501 tgcatgaccc gcaagcccgg tgcctgatct agagggcccg tttaaacccg ctgatcagcc
     1561 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     1621 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     1681 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     1741 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggg tctcgacatt
     1801 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     1861 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     1921 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     1981 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     2041 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     2101 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     2161 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
     2221 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
     2281 cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg
     2341 cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
     2401 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcggggagtc
     2461 gctgcgacgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
     2521 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg
     2581 ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct
     2641 tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg
     2701 tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg
     2761 gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg
     2821 tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt
     2881 gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc
     2941 tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc
     3001 ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc
     3061 gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg
     3121 tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg
     3181 acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc
     3241 tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct
     3301 tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg
     3361 ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg
     3421 cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg
     3481 caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattccgcc accatggtgt
     3541 ctaagggcga agagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg
     3601 tgaacaacca ccacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc
     3661 agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg
     3721 ctaccagctt catgtacggc agcagaacct tcatcaacca cacccagggc atccccgatt
     3781 tctttaagca gtccttccct gagggcttca catgggagag agtcaccaca tacgaagacg
     3841 ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg
     3901 tcaagatcag aggggtgaac ttcccatcca acggccctgt gatgcagaag aaaacactcg
     3961 gctgggaggc caacaccgag atgctgtacc ccgctgacgg cggcctggaa ggcagaaccg
     4021 acatggccct gaagctcgtg ggcgggggcc acctgatctg caacttcaag accacataca
     4081 gatccaagaa acccgctaag aacctcaaga tgcccggcgt ctactatgtg gaccacagac
     4141 tggaaagaat caaggaggcc gacaaagaga cctacgtcga gcagcacgag gtggctgtgg
     4201 ccagatactg cgacctccct agcaaactgg ggcacaaact taatggcatg gacgagctgt
     4261 acaagtccgg actcagatct cgagctcaag cttcgaattc aaagatgagc aaagatggta
     4321 aaaagaagaa aaagaagtca aagacaaagt gtgtaattat gtaaacgcgt gaattcactc
     4381 ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca
     4441 aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg
     4501 agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt
     4561 ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg
     4621 agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt
     4681 ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata
     4741 gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt
     4801 taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac
     4861 tactcccagt catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc
     4921 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4981 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     5041 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5101 cggatcgaca gtactaagct ttactaggga caggattggt gacagaaaag ccccatcctt
     5161 aggcctcctc cttcctagtc tcctgatatt gggtctaacc cccacctcct gttaggcaga
     5221 ttccttatct ggtgacacac ccccatttcc tggagccatc tctctccttg ccagaacctc
     5281 taaggtttgc ttacgatgga gccagagagg atcctgggag ggagagcttg gcagggggtg
     5341 ggagggaagg gggggatgcg tgacctgccc ggttctcagt ggccaccctg cgctaccctc
     5401 tcccagaacc tgagctgctc tgacgcggct gtctggtgcg tttcactgat cctggtgctg
     5461 cagcttcctt acacttccca agaggagaag cagtttggaa aaacaaaatc agaataagtt
     5521 ggtcctgagt tctaactttg gctcttcacc tttctagtcc ccaatttata ttgttcctcc
     5581 gtgcgtcagt tttacctgtg agataaggcc agtagccagc cccgtcctgg cagggctgtg
     5641 gtgaggaggg gggtgtccgt gtggaaaact ccctttgtga gaatggtgcg tcctaggtgt
     5701 tcaccaggtc gtggccgcct ctactccctt tctctttctc catccttctt tccttaaaga
     5761 gtccccagtg ctatctggga catattcctc cgcccagagc agggtcccgc ttccctaagg
     5821 ccctgctctg ggcttctggg tttgagtcct tggcaagccc aggagaggcg ctcaggcttc
     5881 cctgtccccc ttcctcgtcc accatctcat gcccctggct ctcctgcccc ttccctacag
     5941 gggttcctgg ctctgctct
//
