LOCUS       sequence-411117-        7451 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Repair template for the integration of a mNG2(1-10) expression 
            cassette into the AAVS1 locus in human cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7451)
  AUTHORS   Husser MC, Pham NP, Law C, Araujo FRB, Martin VJJ, Piekny A
  TITLE     Endogenous tagging using split mNeonGreen in human iPSCs for live 
            imaging studies.
  JOURNAL   Elife. 2024 Apr 23;12:RP92819. doi: 10.7554/eLife.92819.
  PUBMED    38652106
REFERENCE   2  (bases 1 to 7451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2024-04-23"; volume: "12:RP92819. doi"; pages: " 
            10.7554/eLife.92819"
COMMENT     Sequence Label: pAAVS1-Puro-CAG-mNG2(1-10)
FEATURES             Location/Qualifiers
     source          1..7451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    175..200
                     /label=SA
                     /note="splice acceptor site"
     CDS             224..277
                     /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             287..886
                     /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(287..306)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     783..803
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(918..935)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    924..1148
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        1152..1531
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1809..2825
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2749..2771
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2833..2852
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2880..2889
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3141..3150
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3611..3630)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3676..3731
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3730..3749)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     misc_feature    4105..4941
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     CDS             complement(join(5048..5839,5840..5908))
                     /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     5671..5690
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5909..6013)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6116..6704
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6605..6624
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    join(6816..7451,1..168)
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
ORIGIN
        1 tagtcttctt cctccaaccc gggcccctat gtccacttca ggacagcatg tttgctgcct
       61 ccagggatcc tgtgtccccg agctgggacc accttatatt cccagggccg gttaatgtgg
      121 ctctggttct gggtactttt atctgtcccc tccaccccac agtggggcaa gcttctgacc
      181 tcttctcttc ctcccacagg gcctcgagag atctggcagc ggagagggca gaggaagtct
      241 tctaacatgc ggtgacgtgg aggagaatcc cggccctagg ctcgagatga ccgagtacaa
      301 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc
      361 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg
      421 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
      481 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
      541 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
      601 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
      661 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
      721 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaaa cctccgcgcc
      781 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
      841 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgatcta gagggcccgt
      901 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
      961 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     1021 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     1081 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     1141 ctctatgggt cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     1201 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     1261 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     1321 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     1381 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     1441 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     1501 catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac
     1561 tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt
     1621 ttgtgcagcg atgggggcgg gggggggggg ggggsgsgcs aggggggggg gggggggggg
     1681 aggggggggg gggggsgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc
     1741 gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc
     1801 ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc
     1861 gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc
     1921 cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc
     1981 tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggg
     2041 gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct
     2101 gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg
     2161 cggccggggg cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc
     2221 ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc
     2281 ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgta
     2341 cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggkg ggggtgccgg
     2401 gcggggcggg gccgcckcgg gccggggrgg gctcggggga ggggcgcggc ggcccccgga
     2461 gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg
     2521 agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc
     2581 gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg
     2641 gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg
     2701 gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct
     2761 ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc
     2821 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattcg
     2881 ccaccatggt gagcaagggc gaggaggata acatggcctc tctcccagcg acacatgagt
     2941 tacacatctt tggctccatc aacggtgtgg actttgacat ggtgggtcag ggcaccggca
     3001 atccaaatga tggttatgag gagttaaacc tgaagtccac caagggtgac ctccagttct
     3061 ccccctggat tctggtccct catatcgggt atggcttcca tcagtacctg ccctaccctg
     3121 acgggatgtc gcctttccag gccgccatgg tagatggctc cggataccaa gtccatcgca
     3181 caatgcagtt tgaagatggt gcctccctta ctgttaacta ccgctacacc tacgagggaa
     3241 gccacatcaa aggagaggcc caggtgatgg ggactggttt ccctgctgac ggtcctgtga
     3301 tgaccaacac gctgaccgct gcggactggt gcatgtcgaa gaagacttac cccaacgaca
     3361 aaaccatcat cagtaccttt aagtggagtt acaccactgt aaatggcaag cgctaccgga
     3421 gcactgcgcg gaccacctac acctttgcca agccaatggc ggctaactat ctgaagaacc
     3481 agccgatgta cgtgttccgt aagacggagc tcaagcactc catgtaagaa ttgactcctc
     3541 aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat
     3601 accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc
     3661 atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt
     3721 tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt
     3781 atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc
     3841 tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa
     3901 aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa
     3961 catccctaaa attttcctta catgttttac tagccagatt tttcctcctc tcctgactac
     4021 tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag cccaagcttg
     4081 gatccctcga cagtactaag ctttactagg gacaggattg gtgacagaaa agccccatcc
     4141 ttaggcctcc tccttcctag tctcctgata ttgggtctaa cccccacctc ctgttaggca
     4201 gattccttat ctggtgacac acccccattt cctggagcca tctctctcct tgccagaacc
     4261 tctaaggttt gcttacgatg gagccagaga ggatcctggg agggagagct tggcaggggg
     4321 tgggagggaa gggggggatg cgtgacctgc ccggttctca gtggccaccc tgcgctaccc
     4381 tctcccagaa cctgagctgc tctgacgcgg ctgtctggtg cgtttcactg atcctggtgc
     4441 tgcagcttcc ttacacttcc caagaggaga agcagtttgg aaaaacaaaa tcagaataag
     4501 ttggtcctga gttctaactt tggctcttca cctttctagt ccccaattta tattgttcct
     4561 ccgtgcgtca gttttacctg tgagataagg ccagtagcca gccccgtcct ggcagggctg
     4621 tggtgaggag gggggtgtcc gtgtggaaaa ctccctttgt gagaatggtg cgtcctaggt
     4681 gttcaccagg tcgtggccgc ctctactccc tttctctttc tccatccttc tttccttaaa
     4741 gagtccccag tgctatctgg gacatattcc tccgcccaga gcagggtccc gcttccctaa
     4801 ggccctgctc tgggcttctg ggtttgagtc cttggcaagc ccaggagagg cgctcaggct
     4861 tccctgtccc ccttcctcgt ccaccatctc atgcccctgg ctctcctgcc ccttccctac
     4921 aggggttcct ggctctgctc tccgagcggc cgcgattatc aaaaaggatc ttcacctaga
     4981 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5041 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5101 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5161 ctggccccag tgctgcaatg ataccgcggg acccacgctc accggctcca gatttatcag
     5221 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5281 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5341 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5401 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5461 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5521 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5581 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5641 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5701 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5761 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5821 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5881 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5941 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6001 taggggttcc gcgcacattt ccccgaaaag tgccacctgt catgaccaaa atcccttaac
     6061 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6121 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6181 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6241 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6301 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6361 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6421 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6481 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6541 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6601 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6661 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6721 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6781 cccctgattc tgtggataac cgtgcggccg ccaattgctt tctctgacca gcattctctc
     6841 ccctgggcct gtgccgcttt ctgtctgcag cttgtggcct gggtcacctc tacggctggc
     6901 ccagatcctt ccctgccgcc tccttcaggt tccgtcttcc tccactccct cttccccttg
     6961 ctctctgctg tgttgctgcc caaggatgct ctttccggag cacttccttc tcggcgctgc
     7021 accacgtgat gtcctctgag cggatcctcc ccgtgtctgg gtcctctccg ggcatctctc
     7081 ctccctcacc caaccccatg ccgtcttcac tcgctgggtt cccttttcct tctccttctg
     7141 gggcctgtgc catctctcgt ttcttaggat ggccttctcc gacggatgtc tcccttgcgt
     7201 cccgcctccc cttcttgtag gcctgcatca tcaccgtttt tctggacaac cccaaagtac
     7261 cccgtctccc tggctttagc cacctctcca tcctcttgct ttctttgcct ggacaccccg
     7321 ttctcctgtg gattcgggtc acctctcact cctttcattt gggcagctcc cctacccccc
     7381 ttacctctct agtctgtgct agctcttcca gccccctgtc atggcatctt ccaggggtcc
     7441 gagagctcag c
//
