LOCUS       sequence-362562-        7963 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAVS1 targeting construct with the phiC31 landing pad cassette. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7963)
  AUTHORS   Blanch-Asensio A, Grandela C, Brandao KO, de Korte T, Mei H, 
            Ariyurek Y, Yiangou L, Mol MPH, van Meer BJ, Kloet SL, Mummery CL, 
            Davis RP
  TITLE     STRAIGHT-IN enables high-throughput targeting of large DNA payloads 
            in human pluripotent stem cells.
  JOURNAL   Cell Rep Methods. 2022 Sep 22;2(10):100300. doi: 
            10.1016/j.crmeth.2022.100300. eCollection 2022 Oct 24.
  PUBMED    36313798
REFERENCE   2  (bases 1 to 7963)
  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: "Cell Rep 
            Methods."; date: "2022-09-22"; volume: "2(10):100300. doi"; pages: "
            10.1016/j.crmeth.2022.100300. eCollection 2022 Oct 24"
COMMENT     Sequence Label: AAVS1-phiC31-LP-TC
FEATURES             Location/Qualifiers
     source          1..7963
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    33..836
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     850..866
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    876..923
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        949..1448
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(984..1007)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1381..1400
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1455..1464
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1461..2171
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1606..1624)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1874..1893
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2190..2207)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2196..2420
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             2479..2850
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="AKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDDV
                     TLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGRE
                     FALRDPAGNCVHFVAEEQD"
     polyA_signal    2980..3101
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3017..3036)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3071..3090
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    3117..3150
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     promoter        complement(3170..3188)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    3199..4035
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        complement(4070..4088)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4095..4111)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4249..4551
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4430..4449
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(4954..4973)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5568..5587
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(5948..6739,6740..6808))
                     /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     6571..6590
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6932..7520
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7421..7440
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7674..7691
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7808..7829
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7844..7849,7850..7867,7868..7874)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7882..7898
                     /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     7887..7909
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7906..7922
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7906..7922
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        7943..7961
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ctagtcctgc aggtttaaac gaattcgccc tttgctttct ctgaccagca ttctctcccc
       61 tgggcctgtg ccgctttctg tctgcagctt gtggcctggg tcacctctac ggctggccca
      121 gatccttccc tgccgcctcc ttcaggttcc gtcttcctcc actccctctt ccccttgctc
      181 tctgctgtgt tgctgcccaa ggatgctctt tccggagcac ttccttctcg gcgctgcacc
      241 acgtgatgtc ctctgagcgg atcctccccg tgtctgggtc ctctccgggc atctctcctc
      301 cctcacccaa ccccatgccg tcttcactcg ctgggttccc ttttccttct ccttctgggg
      361 cctgtgccat ctctcgtttc ttaggatggc cttctccgac ggatgtctcc cttgcgtccc
      421 gcctcccctt cttgtaggcc tgcatcatca ccgtttttct ggacaacccc aaagtacccc
      481 gtctccctgg ctttagccac ctctccatcc tcttgctttc tttgcctgga caccccgttc
      541 tcctgtggat tcgggtcacc tctcactcct ttcatttggg cagctcccct acccccctta
      601 cctctctagt ctgtgctagc tcttccagcc ccctgtcatg gcatcttcca ggggtccgag
      661 agctcagcta gtcttcttcc tccaacccgg gcccctatgt ccacttcagg acagcatgtt
      721 tgctgcctcc agggatcctg tgtccccgag ctgggaccac cttatattcc cagggccggt
      781 taatgtggct ctggttctgg gtacttttat ctgtcccctc caccccacag tggggcaagt
      841 cacgacgttg taaaacgacg gccagtctta attaagaagt tcctattccg aagttcctat
      901 tctctagaaa gtataggaac ttcagcttga tatcgaatta attctaccgg gtaggggagg
      961 cgcttttccc aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct
     1021 acacaagtgg cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc
     1081 cgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc
     1141 ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc
     1201 tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc
     1261 aatagcagct ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg
     1321 gcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg
     1381 cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg
     1441 gcctttcgac aattgccacc atggtgagca agggcgagga ggataacatg gccatcatca
     1501 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     1561 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     1621 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     1681 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
     1741 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
     1801 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
     1861 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc
     1921 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     1981 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     2041 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca
     2101 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     2161 tgtacaagta aatcgcgcgc gactcgagcc ctcgactgtg ccttctagtt gccagccatc
     2221 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2281 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2341 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     2401 ggatgcggtg ggctctatgg atccgtagtg ccccaactgg ggtaaccttt gagttctctc
     2461 agttgggggc gtaggcgcgc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac
     2521 gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag
     2581 gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac
     2641 caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac
     2701 gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc
     2761 gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc
     2821 gtgcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc
     2881 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     2941 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     3001 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3061 cattctagtt gtggtttgtc caaactcatc aatgtatctt atgaagttcc tattccgaag
     3121 ttcctattct tcaaatagta taggaacttc gaacatccag ctgatatccc ctatagtgag
     3181 tcgtattaca tggtcattac tagggacagg attggtgaca gaaaagcccc atccttaggc
     3241 ctcctccttc ctagtctcct gatattgggt ctaaccccca cctcctgtta ggcagattcc
     3301 ttatctggtg acacaccccc atttcctgga gccatctctc tccttgccag aacctctaag
     3361 gtttgcttac gatggagcca gagaggatcc tgggagggag agcttggcag ggggtgggag
     3421 ggaagggggg gatgcgtgac ctgcccggtt ctcagtggcc accctgcgct accctctccc
     3481 agaacctgag ctgctctgac gcggctgtct ggtgcgtttc actgatcctg gtgctgcagc
     3541 ttccttacac ttcccaagag gagaagcagt ttggaaaaac aaaatcagaa taagttggtc
     3601 ctgagttcta actttggctc ttcacctttc tagtccccaa tttatattgt tcctccgtgc
     3661 gtcagtttta cctgtgagat aaggccagta gccagccccg tcctggcagg gctgtggtga
     3721 ggaggggggt gtccgtgtgg aaaactccct ttgtgagaat ggtgcgtcct aggtgttcac
     3781 caggtcgtgg ccgcctctac tccctttctc tttctccatc cttctttcct taaagagtcc
     3841 ccagtgctat ctgggacata ttcctccgcc cagagcaggg tcccgcttcc ctaaggccct
     3901 gctctgggct tctgggtttg agtccttggc aagcccagga gaggcgctca ggcttccctg
     3961 tcccccttcc tcgtccacca tctcatgccc ctggctctcc tgccccttcc ctacaggggt
     4021 tcctggctct gctctaaggg cgaattcgcg gccgctaaat tcaattcgcc ctatagtgag
     4081 tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     4141 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     4201 gcccgcaccg atcgcccttc ccaacagttg cgcagcctat acgtacggca gtttaaggtt
     4261 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt
     4321 gacacgccgg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa
     4381 gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc
     4441 accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac
     4501 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc
     4561 atgagattat caaaaaggat cttcacctag atccttttca cgtagaaagc cagtccgcag
     4621 aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca
     4681 agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg
     4741 gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg
     4801 aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga
     4861 tcaagctctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     4921 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     4981 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     5041 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     5101 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     5161 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     5221 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     5281 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     5341 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     5401 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     5461 catgcatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     5521 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     5581 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     5641 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgaat
     5701 tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5761 acaccgcatc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5821 tctaaataca ttcaaatatg tatccgctca tgagattatc aaaaaggatc ttcacctaga
     5881 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5941 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6001 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6061 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6121 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6181 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6241 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6301 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6361 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6421 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6481 gcttttctgt gactggtgaa tactcaacca agtcattctg agaatagtgt atgcggcgac
     6541 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6601 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6661 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6721 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6781 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6841 atcagggtta ttgtctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6901 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6961 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     7021 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     7081 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     7141 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7201 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7261 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7321 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7381 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7441 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7501 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7561 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     7621 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     7681 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     7741 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     7801 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     7861 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     7921 accatgatta cgccaagctc agaattaacc ctcactaaag gga
//
