LOCUS       sequence-431683-        7518 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BxbI landing-pad HDR donor for targeting Olonne-18 GSH. CAGG-driven 
            Casp9-P2A-Bsd..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7518)
  AUTHORS   Autio MI, Motakis E, Perrin A, Bin Amin T, Tiang Z, Do DV, Wang J, 
            Tan JKM, Ding SSL, Tan WX, Lee CJM, Teo AKK, Foo R
  TITLE     Computationally defined and in vitro validated putative genomic safe
            harbour loci for transgene expression in human cells.
  JOURNAL   Elife. 2024 Jan 2;13:e79592. doi: 10.7554/eLife.79592.
  PUBMED    38164941
REFERENCE   2  (bases 1 to 7518)
  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: "2024-01-2"; volume: "13:e79592. doi"; pages: " 
            10.7554/eLife.79592"
COMMENT     Sequence Label: pMIA4.721-Olonne
FEATURES             Location/Qualifiers
     source          1..7518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2772..3151
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          3594..4610
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4534..4556
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4618..4637
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    4797..4844
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             4868..5188
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             complement(5540..5551)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             6065..6091
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6107..6163
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6164..6562
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    6574..6621
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     polyA_signal    6654..6735
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     6679..6698
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(7122..7139)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7122..7138)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7131..7153)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7349..7368)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7468..7490
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ctgggcccag ccggccagat ctgagctcgc ggccgcgatg
     1921 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     1981 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttt caatactgca
     2041 tgagttccgc aaataaacca tcccactcac catcactgtt catgcatttc ctacaacagc
     2101 ctatatctag ttcatccatg tccgcttcta tcttttctct cccaggtttc tgcagcaatc
     2161 atactctcat ttctttctag gggctctaac ttctgcttct ctgcttgtag gcctatgtcc
     2221 ttctcttctg ttaatgacat tgggcttttc cccctcttta tgcacctgca gtcaaggtgg
     2281 gaaacaaaaa gattcgaaac tattttgact cagacctagg aacttgtaat gtcaccataa
     2341 gatgagattc ctgtctccag ccaaatctca ccttgaattg taataatccc tatgtgtcaa
     2401 cggaggagcc aggtagagat aattgaatta taggggtggt ttcccccata ctgttctcat
     2461 ggtagtgaat aagtctcaca agatctgatg gttctataaa tggaagatcc cccgcacaag
     2521 ctctcttgcc tgctaccatg taagacatgc ctttgcttct cctttacctt ctgccatgat
     2581 tgtaaggcct ccccagccag gtggaattgt gagtccatta agcctctttt atatatatat
     2641 aaattaccca gtctcagata tgtctttatt aacagtgtga gaacagacta caacctgacc
     2701 ctgacaatcc ttgtgcttcc ctcatcttgc ctcttctatt tccccactct gcctctgaat
     2761 catcgtcgat cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     2821 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     2881 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     2941 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     3001 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     3061 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     3121 catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac
     3181 tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt
     3241 ttgtgcagcg atgggggcgg gggggggggg ggggggggcc ccgggggggg gggggggggg
     3301 gggggggggg gggggggggg gggggggggg gggggggggg gccccccagg gggggggggg
     3361 gggggggggg gggggcgcgg cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc
     3421 cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc cggggcgggg
     3481 cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
     3541 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg
     3601 ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
     3661 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
     3721 gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg
     3781 aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg
     3841 tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc
     3901 gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg
     3961 ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg
     4021 gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg cacccccctc
     4081 cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg
     4141 ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc
     4201 ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc
     4261 gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac
     4321 ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta
     4381 gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc
     4441 gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg
     4501 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     4561 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     4621 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attctcgagg actgggaaaa
     4681 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     4741 tagcgaagag gcccgcaccg atcgcccttc ccaacagtta attcccgggg atccgtggtt
     4801 tgtctggtca accaccgcgc tctcagtggt gtacggtaca aacccacacg tggccaccat
     4861 gctcgaggga gtgcaggtgg agactatctc cccaggagac gggcgcacct tccccaagcg
     4921 cggccagacc tgcgtggtgc actacaccgg gatgcttgaa gatggaaaga aagttgattc
     4981 ctcccgggac agaaacaagc cctttaagtt tatgctaggc aagcaggagg tgatccgagg
     5041 ctgggaagaa ggggttgccc agatgagtgt gggtcagaga gccaaactga ctatatctcc
     5101 agattatgcc tatggtgcca ctgggcaccc aggcatcatc ccaccacatg ccactctcgt
     5161 cttcgatgtg gagcttctaa aactggaatc tggcggtgga tccggagtcg acggatttgg
     5221 tgatgtcggt gctcttgaga gtttgagggg aaatgcagat ttggcttaca tcctgagcat
     5281 ggagccctgt ggccactgcc tcattatcaa caatgtgaac ttctgccgtg agtccgggct
     5341 ccgcacccgc actggctcca acatcgactg tgagaagttg cggcgtcgct tctcctcgct
     5401 gcatttcatg gtggaggtga agggcgacct gactgccaag aaaatggtgc tggctttgct
     5461 ggagctggcg cggcaggacc acggtgctct ggactgctgc gtggtggtca ttctctctca
     5521 cggctgtcag gccagccacc tgcagttccc aggggctgtc tacggcacag atggatgccc
     5581 tgtgtcggtc gagaagattg tgaacatctt caatgggacc agctgcccca gcctgggagg
     5641 gaagcccaag ctctttttca tccaggcctg tggtggggag cagaaagacc atgggtttga
     5701 ggtggcctcc acttcccctg aagacgagtc ccctggcagt aaccccgagc cagatgccac
     5761 cccgttccag gaaggtttga ggaccttcga ccagctggac gccatatcta gtttgcccac
     5821 acccagtgac atctttgtgt cctactctac tttcccaggt tttgtttcct ggagggaccc
     5881 caagagtggc tcctggtacg ttgagaccct ggacgacatc tttgagcagt gggctcactc
     5941 tgaagacctg cagtccctcc tgcttagggt cgctaatgct gtttcggtga aagggattta
     6001 taaacagatg cctggttgct ttaatttcct ccggaaaaaa cttttcttta aaacatcagt
     6061 cgactatccg tacgacgtac cagactacgc actcgacgga agcggagcca ctaacttctc
     6121 cctgttgaaa caagcagggg atgtcgaaga gaatcccggg ccaatggcca agcctttgtc
     6181 tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc
     6241 tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt
     6301 caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc
     6361 tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat
     6421 cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc
     6481 catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc
     6541 tggttatgtg tgggagggct aataaggtac cgtggtttgt ctggtcaacc accgcggtct
     6601 cagtggtgta cggtacaaac ccaaccggtg tttggacaaa ccacaactag aatgcagtga
     6661 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     6721 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     6781 gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaatta gagagaggaa
     6841 tgcttctggg gtaactgcta attgaaagta ggttagtctc ttccacatag cgagagctaa
     6901 tcacccatga tccacaattc caggagcatg aacatgtatc tccctcacca gcctgatgcc
     6961 tgaaattcaa atgcaagaga ttcattcctt gaaattagat attggctcta aagattcttt
     7021 taaagtggga ggtggccaga atgagatgat tgctatagtc aggtttaaca atcaactgaa
     7081 ataccccaga atctgaaaag gttacctcat gcaagcttgg cactggccgt cgttttacaa
     7141 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     7201 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     7261 agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     7321 tcacaccgca tatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7381 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     7441 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     7501 catcaccgaa acgcgcga
//
