LOCUS       sequence-362557-        4615 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  phiC31 landing pad cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4615)
  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 4615)
  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: pENTR-mCherry-attP(C31)-*BleoR
FEATURES             Location/Qualifiers
     source          1..4615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     95..112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(271..298)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(390..476)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     525..547
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     539..556
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     540..556
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    566..613
                     /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        639..1138
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(674..697)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1071..1090
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1145..1154
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1151..1861
                     /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(1296..1314)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1564..1583
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1880..1897)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1886..2110
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             2169..2540
                     /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    2670..2791
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2707..2726)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2761..2780
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2807..2840
                     /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)"
     primer_bind     complement(2859..2878)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2860..2878)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2883..2899)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2883..2899)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3001..3020)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3012..3821
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3079..3098)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3968..4556
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4457..4476
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
       61 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
      121 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
      181 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac
      241 cgctagccag gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc
      301 ttagtttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc
      361 ttcacaacgt tcaaatccgc cgggagcgga tttgtcctac tcaggagagc gttcaccgac
      421 aaacaacaga taaaacgaaa ggcccagtct tccgactgag cctttcgttt tatttgatgc
      481 ctggcagttc cctactctcg cgttaacgct agcatggatg ttttcccagt cacgacgttg
      541 taaaacgacg gccagtctta attaagaagt tcctattccg aagttcctat tctctagaaa
      601 gtataggaac ttcagcttga tatcgaatta attctaccgg gtaggggagg cgcttttccc
      661 aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg
      721 cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg
      781 tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca
      841 gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat
      901 ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct
      961 ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag
     1021 gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac
     1081 gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac
     1141 aattgccacc atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat
     1201 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     1261 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
     1321 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
     1381 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     1441 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     1501 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     1561 cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
     1621 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     1681 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     1741 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
     1801 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta
     1861 aatcgcgcgc gactcgagcc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     1921 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     1981 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2041 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2101 ggctctatgg atccgtagtg ccccaactgg ggtaaccttt gagttctctc agttgggggc
     2161 gtaggcgcgc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag
     2221 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg
     2281 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc
     2341 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt
     2401 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg
     2461 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg
     2521 tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc gccttctatg
     2581 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     2641 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     2701 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2761 gtggtttgtc caaactcatc aatgtatctt atgaagttcc tattccgaag ttcctattct
     2821 tcaaatagta taggaacttc gaacatccag ctgatatccc ctatagtgag tcgtattaca
     2881 tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg
     2941 cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata
     3001 caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca
     3061 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     3121 caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     3181 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     3241 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     3301 ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     3361 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     3421 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     3481 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     3541 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     3601 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     3661 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     3721 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     3781 ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt
     3841 tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat
     3901 cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3961 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4021 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4081 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4141 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4201 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4261 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4321 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4381 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4441 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4501 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4561 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacat
//
