LOCUS       sequence-215897-        4717 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Homology arms and linker-mEGFP sequence for internal insertion of 
            PGK-mEGFP at the AAVS1 safe harbor location in human cells as a 
            reporter for cytoplasmic mEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4717)
  AUTHORS   Allen Institute for Cell Science
  TITLE     Allen Institute for Cell Science Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4717)
  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            "Unpublished"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-36: AAVS1-mEGFP (PGK)
FEATURES             Location/Qualifiers
     source          1..4717
                     /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"
     promoter        1881..2381
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2300..2318
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(2403..2405,2406..2408,2409..3122)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2448..2469)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2709..2728)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3056..3077
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3212..3231)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3277..3332
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3331..3350)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3693..3709)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3693..3709)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3706..3728)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3717..3733
                     /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(3741..3747,3748..3765,3766..3771))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3786..3807
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    3881..4717
                     /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 cgttttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac
     1921 gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat
     1981 tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg
     2041 cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga
     2101 cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag
     2161 caatggcagc gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg
     2221 ccgagagcag cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg
     2281 ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag
     2341 tcggctccct cgttgaccga atcaccgacc tctctcccca gggggatcca ccggagctta
     2401 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     2461 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     2521 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     2581 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     2641 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     2701 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2761 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2821 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     2881 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     2941 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     3001 actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg
     3061 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     3121 agacgcgtga attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg
     3181 ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg
     3241 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
     3301 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc
     3361 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg
     3421 ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg
     3481 tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt
     3541 ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat
     3601 ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc
     3661 tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3721 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3781 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3841 ggaaacctgt cgtgccagcg gatcgacagt actaagcttt actagggaca ggattggtga
     3901 cagaaaagcc ccatccttag gcctcctcct tcctagtctc ctgatattgg gtctaacccc
     3961 cacctcctgt taggcagatt ccttatctgg tgacacaccc ccatttcctg gagccatctc
     4021 tctccttgcc agaacctcta aggtttgctt acgatggagc cagagaggat cctgggaggg
     4081 agagcttggc agggggtggg agggaagggg gggatgcgtg acctgcccgg ttctcagtgg
     4141 ccaccctgcg ctaccctctc ccagaacctg agctgctctg acgcggctgt ctggtgcgtt
     4201 tcactgatcc tggtgctgca gcttccttac acttcccaag aggagaagca gtttggaaaa
     4261 acaaaatcag aataagttgg tcctgagttc taactttggc tcttcacctt tctagtcccc
     4321 aatttatatt gttcctccgt gcgtcagttt tacctgtgag ataaggccag tagccagccc
     4381 cgtcctggca gggctgtggt gaggaggggg gtgtccgtgt ggaaaactcc ctttgtgaga
     4441 atggtgcgtc ctaggtgttc accaggtcgt ggccgcctct actccctttc tctttctcca
     4501 tccttctttc cttaaagagt ccccagtgct atctgggaca tattcctccg cccagagcag
     4561 ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt tgagtccttg gcaagcccag
     4621 gagaggcgct caggcttccc tgtccccctt cctcgtccac catctcatgc ccctggctct
     4681 cctgcccctt ccctacaggg gttcctggct ctgctct
//
