LOCUS       sequence-362559-        4609 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1 landing pad cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4609)
  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 4609)
  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-eGFP-attP(bxb)-*BsdR
FEATURES             Location/Qualifiers
     source          1..4609
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     101..118
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(277..304)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(396..482)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     531..553
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     545..562
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     546..562
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(584..616)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        672..1171
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(707..730)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1104..1123
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1180..1189
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1186..1188,1189..1191,1192..1905)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1186..1188,1189..1191,1192..1902)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1231..1252)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1492..1511)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1839..1860
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1948..2069
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1985..2004)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2039..2058
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2088..2135
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             2141..2536
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    2584..2808
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2819..2851)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     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 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
       61 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      121 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      181 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatac
      241 gcgtaccgct agccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc
      301 ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc
      361 cgttgcttca caacgttcaa atccgctccc ggcggatttg tcctactcag gagagcgttc
      421 accgacaaac aacagataaa acgaaaggcc cagtcttccg actgagcctt tcgttttatt
      481 tgatgcctgg cagttcccta ctctcgcgtt aacgctagca tggatgtttt cccagtcacg
      541 acgttgtaaa acgacggcca gtcgctagcg agctcggcgc gccataactt cgtatagcat
      601 acattatacg aagttagaat tcgccttctg caggagcgta cagaacccag ggccctggca
      661 cccgtgctac cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc
      721 agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc
      781 caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttccactcc
      841 tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat
      901 ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg
      961 ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag
     1021 gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc
     1081 ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct
     1141 gttctcctct tcctcatctc cgggcctttc gacctgcagg ccaccatggt gagcaagggc
     1201 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1261 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1321 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1381 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1441 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1501 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1561 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1621 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1681 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1741 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1801 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1861 accgccgccg ggatcactct cggcatggac gagctgtaca agtaactgca gcgcggggat
     1921 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     1981 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2041 ggtttgtcca aactcatcaa tgtatcttat catgtctaga tatacgtggt ttgtctggtc
     2101 aaccaccgcg gtctcagtgg tgtacggtac aaacccatat gccaagcctt tgtctcaaga
     2161 agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga
     2221 ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt
     2281 atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc
     2341 ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag
     2401 cccctgcgga cggtgtcgac aggtgcttct cgatctgcat cctgggatca aagcgatagt
     2461 gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta
     2521 tgtgtgggag ggctaaatta ccctgttatc cctacctcga ctgtgccttc tactcagcct
     2581 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     2641 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     2701 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     2761 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcat
     2821 aacttcgtat aggagacttt atacgaagtt aatggatccc 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 gccttttgc
//
