LOCUS       sequence-331135-        6261 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAVS1 targeting repair template.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6261)
  AUTHORS   Sun D, Evans L, Perrone F, Sokleva V, Lim K, Rezakhani S, Lutolf M, 
            Zilbauer M, Rawlins EL
  TITLE     A functional genetic toolbox for human tissue-derived organoids.
  JOURNAL   eLife. 2021 Oct 6;10. pii: 67886. doi: 10.7554/eLife.67886.
  PUBMED    34612202
REFERENCE   2  (bases 1 to 6261)
  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: "eLife."; 
            date: "2021-10-6"; volume: "10. pii: 67886. doi"; pages: " 
            10.7554/eLife.67886"
COMMENT     Sequence Label: AAVS1-EF1a-mTagRFP-T-CAAX
FEATURES             Location/Qualifiers
     source          1..6261
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    1212..1713
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        1878..2089
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2102..2370
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      2398..2407
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2404..3135
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     primer_bind     complement(3324..3343)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3389..3444
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3443..3462)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     misc_feature    3778..4614
                     /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(4636..4653)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4636..4652)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4645..4667)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4861..4880)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4980..5002
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5040..5058)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5126..5230
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5231..5299,5300..6091)
                     /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     complement(5449..5468)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctcg gcgcgtattg
     1021 ggatcccttt gctttctctg accagcattc tctcccctgg gcctgtgccg ctttctgtct
     1081 gcagcttgtg gcctgggtca cctctacggc tggcccagat ccttccctgc cgcctccttc
     1141 aggttccgtc ttcctccact ccctcttccc cttgctctct gctgtgttgc tgcccaagga
     1201 tgctctttcc ggagcacttc cttctcggcg ctgcaccacg tgatgtcctc tgagcggatc
     1261 ctccccgtgt ctgggtcctc tccgggcatc tctcctccct cacccaaccc catgccgtct
     1321 tcactcgctg ggttcccttt tccttctcct tctggggcct gtgccatctc tcgtttctta
     1381 ggatggcctt ctccgacgga tgtctccctt gcgtcccgcc tccccttctt gtaggcctgc
     1441 atcatcaccg tttttctgga caaccccaaa gtaccccgtc tccctggctt tagccacctc
     1501 tccatcctct tgctttcttt gcctggacac cccgttctcc tgtggattcg ggtcacctct
     1561 cactcctttc atttgggcag ctcccctacc ccccttacct ctctagtctg tgctagctct
     1621 tccagccccc tgtcatggca tcttccaggg gtccgagagc tcagctagtc ttcttcctcc
     1681 aacccgggcc cctatgtcca cttcaggaca gcatgtttgc tgcctccagg gatcctgtgt
     1741 ccccgagctg ggaccacctt atattcccag ggccggttaa tgtggctctg gttctgggta
     1801 cttttatctg tcccctccac cccacagtgg gccaagctta ctagtaagga tctgcgatcg
     1861 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
     1921 aggggtcggc aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
     1981 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
     2041 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg
     2101 aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg
     2161 gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta
     2221 ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc
     2281 tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt
     2341 ttcgttttct gttctgcgcc gttacagatc caagctgtga ccggcgccta cgaattcgcc
     2401 accatggtgt ctaagggcga agagctgatt aaggagaaca tgcacatgaa gctgtacatg
     2461 gagggcaccg tgaacaacca ccacttcaag tgcacatccg agggcgaagg caagccctac
     2521 gagggcaccc agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc
     2581 gacatcctgg ctaccagctt catgtacggc agcagaacct tcatcaacca cacccagggc
     2641 atccccgatt tctttaagca gtccttccct gagggcttca catgggagag agtcaccaca
     2701 tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc
     2761 atctacaacg tcaagatcag aggggtgaac ttcccatcca acggccctgt gatgcagaag
     2821 aaaacactcg gctgggaggc caacaccgag atgctgtacc ccgctgacgg cggcctggaa
     2881 ggcagaaccg acatggccct gaagctcgtg ggcgggggcc acctgatctg caacttcaag
     2941 accacataca gatccaagaa acccgctaag aacctcaaga tgcccggcgt ctactatgtg
     3001 gaccacagac tggaaagaat caaggaggcc gacaaagaga cctacgtcga gcagcacgag
     3061 gtggctgtgg ccagatactg cgacctccct agcaaactgg ggcacaaact taatggcatg
     3121 gacgagctgt acaagtccgg actcagatct cgagctcaag cttcgaattc aaagatgagc
     3181 aaagatggta aaaagaagaa aaagaagtca aagacaaagt gtgtaattat gtaaacgcgt
     3241 gaattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc
     3301 ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg
     3361 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
     3421 tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa
     3481 catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg
     3541 aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc
     3601 ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta
     3661 tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc
     3721 ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc aagctttact
     3781 agggacagga ttggtgacag aaaagcccca tccttaggcc tcctccttcc tagtctcctg
     3841 atattgggtc taacccccac ctcctgttag gcagattcct tatctggtga cacaccccca
     3901 tttcctggag ccatctctct ccttgccaga acctctaagg tttgcttacg atggagccag
     3961 agaggatcct gggagggaga gcttggcagg gggtgggagg gaaggggggg atgcgtgacc
     4021 tgcccggttc tcagtggcca ccctgcgcta ccctctccca gaacctgagc tgctctgacg
     4081 cggctgtctg gtgcgtttca ctgatcctgg tgctgcagct tccttacact tcccaagagg
     4141 agaagcagtt tggaaaaaca aaatcagaat aagttggtcc tgagttctaa ctttggctct
     4201 tcacctttct agtccccaat ttatattgtt cctccgtgcg tcagttttac ctgtgagata
     4261 aggccagtag ccagccccgt cctggcaggg ctgtggtgag gaggggggtg tccgtgtgga
     4321 aaactccctt tgtgagaatg gtgcgtccta ggtgttcacc aggtcgtggc cgcctctact
     4381 ccctttctct ttctccatcc ttctttcctt aaagagtccc cagtgctatc tgggacatat
     4441 tcctccgccc agagcagggt cccgcttccc taaggccctg ctctgggctt ctgggtttga
     4501 gtccttggca agcccaggag aggcgctcag gcttccctgt cccccttcct cgtccaccat
     4561 ctcatgcccc tggctctcct gccccttccc tacaggggtt cctggctctg ctctatccca
     4621 atggcgcgtc aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     4681 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     4741 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     4801 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
     4861 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     4921 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4981 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     5041 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     5101 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5161 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5221 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5281 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5341 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5401 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5461 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5521 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5581 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5641 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5701 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5761 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5821 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5881 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     5941 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6001 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6061 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6121 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6181 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6241 agaaaagatc aaaggatctt c
//
