LOCUS       sequence-380483-        8532 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CLYBL targeting construct with the Bxb1 landing pad version 2 
            cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8532)
  AUTHORS   Blanch-Asensio A, van der Vaart B, Vinagre M, Groen E, Arendzen C, 
            Freund C, Geijsen N, Mummery CL, Davis RP
  TITLE     Generation of AAVS1 and CLYBL STRAIGHT-IN v2 acceptor human iPSC 
            lines for integrating DNA payloads.
  JOURNAL   Stem Cell Res. 2023 Feb;66:102991. doi: 10.1016/j.scr.2022.102991. 
            Epub 2022 Dec 5.
  PUBMED    36495703
REFERENCE   2  (bases 1 to 8532)
  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: "Stem Cell 
            Res."; date: "2023-02"; volume: "66:102991. doi"; pages: " 
            10.1016/j.scr.2022.102991. Epub 2022 Dec 5"
COMMENT     Sequence Label: CLYBL-Bxb1-LP-v2-TC
FEATURES             Location/Qualifiers
     source          1..8532
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    51..75
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     primer_bind     995..1012
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     996..1012
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(1034..1066)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1122..1621
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1157..1180)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1554..1573
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1630..1639
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1636..1638,1639..1641,1642..2355)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(1681..1702)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    2405..2526
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2442..2461)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2496..2515
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2545..2592
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             2598..2969
                     /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"
     primer_bind     complement(3052..3071)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3117..3172
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3171..3190)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    complement(3560..3592)
                     /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)."
     misc_feature    4452..4476
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      4564..5274
                     /label=oriV
                     /note="incP origin of replication"
     promoter        5295..5399
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5400..6194
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      6365..6953
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6854..6873
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             7289..8437
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
ORIGIN
        1 agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg tggcaggata
       61 tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg gacgttttta
      121 atgtactggg gttgaacact ctgtgccagc aggagaatca tgccaatggg ccaatataca
      181 ttctgaccca cagtttcata ataaaataaa atggttgtgg ttgtaagcca ctatgtttca
      241 gagtggtttg ttacacagca ataaataact aatatagtag gcataccatc aagtccaaag
      301 taggtagaga agaatgtaaa tagcagagca aaacagcatg actggtggct gggaggctta
      361 aaactgggac aggatcagag tcatgaaaga agtcaaagaa atggttcaga agtaaggctg
      421 agactgactt acaaaagctg aaagtccctt taagttggtg tttggtgcat tggcaggggc
      481 aggtatggtg acttaaaaga gccatgctca acaagatcaa gcacaacaca atcacgggtc
      541 accccagcag accttagcga gtctagccat ttctttggtg gtggtcacag tcatgcttca
      601 gcccagtttc cacttggaca aatggtacat attttcaatg agatgaaaat taagatacaa
      661 tccatgtgct cagagagtga tcacagctct gactaaacac tgtgccccaa agtgttgagg
      721 aattgggaaa acctagctga gttagtggtc tcttttctgt tacaataaag ctcataatga
      781 aaattagcct tctttgttct tccccaagtc tttctttcta gacgaaacta ctttcaactg
      841 ttttaacttc cttactgtta acttccatat ttctagatag tatggtcaga ctgttatttc
      901 ttgactttta aatgtaagat attatctact gacttccttc tatgtaagat gaggattgag
      961 ctctcttacc cttctcccaa ctaagtcacg acgttgtaaa acgacggcca gtcgctagcg
     1021 agctcggcgc gccataactt cgtatagcat acattatacg aagttagaat tcgccttctg
     1081 caggagcgta cagaacccag ggccctggca cccgtgctac cgggtagggg aggcgctttt
     1141 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag
     1201 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     1261 tggtggcccc ttcgcgccac cttccactcc tcccctagtc aggaagttcc cccccgcccc
     1321 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     1381 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     1441 gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct
     1501 caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg
     1561 cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc
     1621 gacctgcagg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     1681 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgagggg cgagggcgag
     1741 ggcgatgcca ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1801 gtgccctggc ccaccctcgt gaccaccctg agccacggcg tgcagtgctt cgcccgctac
     1861 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1921 gagcgcacca tcttcttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc
     1981 gagggcgaca ccctagtgaa ccgcatcgag ctgaagggcg tcgacttcaa ggaggacggc
     2041 aacatcctgg ggcacaagct ggagtacaac ttcaacagcc acaacatcta tatcatggcc
     2101 gtcaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacgt ggaggacggc
     2161 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2221 ctgcccgaca gccactacct gagcacccag tccgtgctga gcaaagaccc caacgagaag
     2281 cgcgatcaca tggtcctgct ggagttccgc accgccgccg ggatcactct cggcatggac
     2341 gagctgtaca agtaatcgat aactgcagcg cggggatctc atgctggagt tcttcgccca
     2401 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     2461 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     2521 atcttatcat gtctagatat acgtggtttg tctggtcaac caccgcggtc tcagtggtgt
     2581 acggtacaaa cccatatgcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg
     2641 tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg
     2701 acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc
     2761 aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg
     2821 ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg
     2881 agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg
     2941 tgcacttcgt ggccgaggag caggactgac attcactcct caggtgcagg ctgcctatca
     3001 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc
     3061 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
     3121 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     3181 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     3241 aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta
     3301 tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta
     3361 gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt
     3421 acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc
     3481 tcttctctta tgaagatcgg atcgctacta gaggatgcac atgtgaccga gggaggagct
     3541 gcaagaactc ttcctcacga taacttcgta taggagactt tatacgaagt taagcgcttc
     3601 gtgagaccac gaagtagtcc tcctcatcct tccaacataa atatattttg ggattatatc
     3661 aacattcaat gttacttaaa gtgaccttgt aaatattttc acaactgagc catgtttgat
     3721 ttgtatactt atgtttactt tactgttttt cctgaagtta ataattgcct tgaatttatt
     3781 tatttcttta aaaatgtttc attactcagg actgtagttt acattacgat tctttgtgtt
     3841 atacagttga tgggtttctt ttctttctta atttctttaa aaaatagaga tggggtctta
     3901 ctatattacc caggctggtc ttgaagtcct gggctcaagt gatcttcctg tctcagccta
     3961 ccaagtagct gagactatag gtgcaaaaaa gccactatac ctggctagtt tacaggtttt
     4021 aacaaatgca ttatgccacg tatccattat tacaggatca cacaagatat tttcattacc
     4081 ctgaaagcat ccctgtgttc caccaattca tcctgcctcc atgagccgct ggcaaccact
     4141 gatctctata gttttgcctt ttctaaaatg tcatataatt ggaatcatac agtctgtagc
     4201 attttcagac tagcttttaa aatttggcaa tatgcattta aggttcctcc ttaaatgtga
     4261 agggcatagc caatgtggct tgatagctca tttcttttta ttggtgaata tttcattgtc
     4321 tggatgttcc acagtttgtt tatccattca cctattcaat ttgctttttt tctgtgtatc
     4381 tatcactaat tcaatactgg actctccaac agagccggga cacgaagtga tccgtttaaa
     4441 ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag cgtttattag
     4501 aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgcc
     4561 agccgccttt gcgacgctca ccgggctggt tgccctcgcc gctgggctgg cggccgtcta
     4621 tggccctgca aacgcgccag aaacgccgtc gaagccgtgt gcgagacacc gcggccgccg
     4681 gcgttgtgga tacctcgcgg aaaacttggc cctcactgac agatgagggg cggacgttga
     4741 cacttgaggg gccgactcac ccggcgcggc gttgacagat gaggggcagg ctcgatttcg
     4801 gccggcgacg tggagctggc cagcctcgca aatcggcgaa aacgcctgat tttacgcgag
     4861 tttcccacag atgatgtgga caagcctggg gataagtgcc ctgcggtatt gacacttgag
     4921 gggcgcgact actgacagat gaggggcgcg atccttgaca cttgaggggc agagtgctga
     4981 cagatgaggg gcgcacctat tgacatttga ggggctgtcc acaggcagaa aatccagcat
     5041 ttgcaagggt ttccgcccgt ttttcggcca ccgctaacct gtcttttaac ctgcttttaa
     5101 accaatattt ataaaccttg tttttaacca gggctgcgcc ctgtgcgcgt gaccgcgcac
     5161 gccgaagggg ggtgcccccc cttctcgaac cctcccggcc cgctaacgcg ggcctcccat
     5221 ccccccaggg gctgcgcccc tcggccgcga acggcctcac cccaaaaatg gcagcgctgg
     5281 ccaattcccg agtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5341 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5401 tggctaaaat gagaatatca ccggaattga aaaaactgat cgaaaaatac cgctgcgtaa
     5461 aagatacgga aggaatgtct cctgctaagg tatataagct ggtgggagaa aatgaaaacc
     5521 tatatttaaa aatgacggac agccggtata aagggaccac ctatgatgtg gaacgggaaa
     5581 aggacatgat gctatggctg gaaggaaagc tgcctgttcc aaaggtcctg cactttgaac
     5641 ggcatgatgg ctggagcaat ctgctcatga gtgaggccga tggcgtcctt tgctcggaag
     5701 agtatgaaga tgaacaaagc cctgaaaaga ttatcgagct gtatgcggag tgcatcaggc
     5761 tctttcactc catcgacata tcggattgtc cctatacgaa tagcttagac agccgcttag
     5821 ccgaattgga ttacttactg aataacgatc tggccgatgt ggattgcgaa aactgggaag
     5881 aggacactcc atttaaagat ccgcgcgagc tgtatgattt tttaaagacg gaaaagcccg
     5941 aagaggaact tgtcttttcc cacggcgacc tgggagacag caacatcttt gtgaaagatg
     6001 gcaaagtaag tggctttatt gatcttggga gaagcggcag ggcggacaag tggtatgaca
     6061 ttgccttctg cgtccggtcg ctcagggagg atatcgggga agaacagtat gtcgagctat
     6121 tttttgactt actggggatc aagcctgatt gggagaaaat aaaatattat attttactgg
     6181 atgaattgtt ttagctgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6241 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6301 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6361 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6421 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6481 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6541 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6601 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6661 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6721 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6781 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6841 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6901 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6961 gcaacgcggc ctttttacgg ttcctggcag atcctagatg tggcgcaacg atgccggcga
     7021 caagcaggag cgcaccgact tcttccgcat caagtgtttt ggctctcagg ccgaggccca
     7081 cggcaagtat ttgggcaagg ggtcgctggt attcgtgcag ggcaagattc ggaataccaa
     7141 gtacgagaag gacggccaga cggtctacgg gaccgacttc attgccgata aggtggatta
     7201 tctggacacc aaggcaccag gcgggtcaaa tcaggaataa gggcacattg ccccggcgtg
     7261 agtcggggca atcccgcaag gagggtgaat gaatcggacg tttgaccgga aggcatacag
     7321 gcaagaactg atcgacgcgg ggttttccgc cgaggatgcc gaaaccatcg caagccgcac
     7381 cgtcatgcgt gcgccccgcg aaaccttcca gtccgtcggc tcgatggtcc agcaagctac
     7441 ggccaagatc gagcgcgaca gcgtgcaact ggctccccct gccctgcccg cgccatcggc
     7501 cgccgtggag cgttcgcgtc gtcttgaaca ggaggcggca ggtttggcga agtcgatgac
     7561 catcgacacg cgaggaacta tgacgaccaa gaagcgaaaa accgccggcg aggacctggc
     7621 aaaacaggtc agcgaggcca agcaggccgc gttgctgaaa cacacgaagc agcagatcaa
     7681 ggaaatgcag ctttccttgt tcgatattgc gccgtggccg gacacgatgc gagcgatgcc
     7741 aaacgacacg gcccgctctg ccctgttcac cacgcgcaac aagaaaatcc cgcgcgaggc
     7801 gctgcaaaac aaggtcattt tccacgtcaa caaggacgtg aagatcacct acaccggcgt
     7861 cgagctgcgg gccgacgatg acgaactggt gtggcagcag gtgttggagt acgcgaagcg
     7921 cacccctatc ggcgagccga tcaccttcac gttctacgag ctttgccagg acctgggctg
     7981 gtcgatcaat ggccggtatt acacgaaggc cgaggaatgc ctgtcgcgcc tacaggcgac
     8041 ggcgatgggc ttcacgtccg accgcgttgg gcacctggaa tcggtgtcgc tgctgcaccg
     8101 cttccgcgtc ctggaccgtg gcaagaaaac gtcccgttgc caggtcctga tcgacgagga
     8161 aatcgtcgtg ctgtttgctg gcgaccacta cacgaaattc atatgggaga agtaccgcaa
     8221 gctgtcgccg acggcccgac ggatgttcga ctatttcagc tcgcaccggg agccgtaccc
     8281 gctcaagctg gaaaccttcc gcctcatgtg cggatcggat tccacccgcg tgaagaagtg
     8341 gcgcgagcag gtcggcgaag cctgcgaaga gttgcgaggc agcggcctgg tggaacacgc
     8401 ctgggtcaat gatgacctgg tgcattgcaa acgctagggc cttgtggggt cagttccggc
     8461 tgggggttca gcagcccctg ctcggatctg ttggaccgga cagtagtcat ggttgatggg
     8521 ctgcctgtat cg
//
