LOCUS       sequence-431682-        7050 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BxbI landing-pad HDR donor for targeting Keppel-19 GSH. CAGG-driven 
            Casp9-P2A-Bsd..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7050)
  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 7050)
  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-Keppel
FEATURES             Location/Qualifiers
     source          1..7050
                     /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        2243..2622
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2624..2901
                     /label=chicken beta-actin promoter
     intron          2902..3918
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3842..3864
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3926..3945
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    4105..4152
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             4176..4496
                     /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(4848..4859)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             5373..5399
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5415..5471
                     /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             5472..5870
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    5882..5929
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     polyA_signal    5962..6043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5987..6006
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(6654..6671)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6654..6670)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6663..6685)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6881..6900)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7000..7022
                     /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 ccaacagtta ggagccagaa
     2041 gtgcaaagcg ccaggagttt ccaagcagag aggacagcac gtaccaagga ccagtggtgg
     2101 gaatgagctt ggagacacgg acacacagaa tcagagacag accgaaacaa agagacagag
     2161 acaggcgctc agggagaccc taacagagac agacagacaa agagacaagg gtctctcatt
     2221 ggccggcata tcatcgtcga tcgacattga ttattgacta gttattaata gtaatcaatt
     2281 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     2341 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     2401 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     2461 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     2521 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     2581 acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg
     2641 ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt
     2701 ttttaattat tttgtgcagc gatgggggcg gggggggggg gggggcgcgc gccaggcggg
     2761 gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag
     2821 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
     2881 aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc
     2941 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
     3001 gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt
     3061 tcttttctgt ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg
     3121 agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg
     3181 ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg
     3241 cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac
     3301 aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc
     3361 gggctgcaac cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt
     3421 gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag
     3481 gtgggggtgc cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc
     3541 ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta
     3601 tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctgtgc ggagccgaaa
     3661 tctgggaggc gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc
     3721 aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct
     3781 ctccagcctc ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg
     3841 gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc
     3901 ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt
     3961 ggcaaagaat tctcgaggac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     4021 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     4081 aacagttaat tcccggggat ccgtggtttg tctggtcaac caccgcgctc tcagtggtgt
     4141 acggtacaaa cccacacgtg gccaccatgc tcgagggagt gcaggtggag actatctccc
     4201 caggagacgg gcgcaccttc cccaagcgcg gccagacctg cgtggtgcac tacaccggga
     4261 tgcttgaaga tggaaagaaa gttgattcct cccgggacag aaacaagccc tttaagttta
     4321 tgctaggcaa gcaggaggtg atccgaggct gggaagaagg ggttgcccag atgagtgtgg
     4381 gtcagagagc caaactgact atatctccag attatgccta tggtgccact gggcacccag
     4441 gcatcatccc accacatgcc actctcgtct tcgatgtgga gcttctaaaa ctggaatctg
     4501 gcggtggatc cggagtcgac ggatttggtg atgtcggtgc tcttgagagt ttgaggggaa
     4561 atgcagattt ggcttacatc ctgagcatgg agccctgtgg ccactgcctc attatcaaca
     4621 atgtgaactt ctgccgtgag tccgggctcc gcacccgcac tggctccaac atcgactgtg
     4681 agaagttgcg gcgtcgcttc tcctcgctgc atttcatggt ggaggtgaag ggcgacctga
     4741 ctgccaagaa aatggtgctg gctttgctgg agctggcgcg gcaggaccac ggtgctctgg
     4801 actgctgcgt ggtggtcatt ctctctcacg gctgtcaggc cagccacctg cagttcccag
     4861 gggctgtcta cggcacagat ggatgccctg tgtcggtcga gaagattgtg aacatcttca
     4921 atgggaccag ctgccccagc ctgggaggga agcccaagct ctttttcatc caggcctgtg
     4981 gtggggagca gaaagaccat gggtttgagg tggcctccac ttcccctgaa gacgagtccc
     5041 ctggcagtaa ccccgagcca gatgccaccc cgttccagga aggtttgagg accttcgacc
     5101 agctggacgc catatctagt ttgcccacac ccagtgacat ctttgtgtcc tactctactt
     5161 tcccaggttt tgtttcctgg agggacccca agagtggctc ctggtacgtt gagaccctgg
     5221 acgacatctt tgagcagtgg gctcactctg aagacctgca gtccctcctg cttagggtcg
     5281 ctaatgctgt ttcggtgaaa gggatttata aacagatgcc tggttgcttt aatttcctcc
     5341 ggaaaaaact tttctttaaa acatcagtcg actatccgta cgacgtacca gactacgcac
     5401 tcgacggaag cggagccact aacttctccc tgttgaaaca agcaggggat gtcgaagaga
     5461 atcccgggcc aatggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa
     5521 cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc gcagctctct
     5581 ctagcgacgg ccgcatcttc actggtgtca atgtatatca ttttactggg ggaccttgtg
     5641 cagaactcgt ggtgctgggc actgctgctg ctgcggcagc tggcaacctg acttgtatcg
     5701 tcgcgatcgg aaatgagaac aggggcatct tgagcccctg cggacggtgc cgacaggtgc
     5761 ttctcgatct gcatcctggg atcaaagcca tagtgaagga cagtgatgga cagccgacgg
     5821 cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctaa taaggtaccg
     5881 tggtttgtct ggtcaaccac cgcggtctca gtggtgtacg gtacaaaccc aaccggtgtt
     5941 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     6001 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     6061 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     6121 ctacaaatgt ggtaatatgg gttacatcgt ctctccccca aaaaacctat atgctgaagt
     6181 cctcacccct cagaacctcc gaatgtactg gaggtggtac agatgtaact ggagtagggt
     6241 gggctctaat ctaatacgac tgatgacctt ataaaagggg acaatgtggt tcatgcctgt
     6301 aatcccagca ctttgggagg ctgaggcggg ggggatcact tgaggtcagg agttcgagac
     6361 cagcctggcc aacatggcaa aaccctgtct ctactaaaaa tagaaaaatt agctgggtgt
     6421 ggtcgtgggc gcctgtaatc caagctactc cagaggctga ggcaggagaa tcccttgaac
     6481 ctgggaggcg gaggttgcag tgagctgaaa tcgtgccatt gctctccagc ctgggcaaca
     6541 gagtgagaag attccttctc aaaacaaaca aaaaaacgaa ggggcagggg gacaatgtgg
     6601 gcacaggtat gcgtgcaggg agaatgctgt gtgaatatgc atgcaagctt ggcactggcc
     6661 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     6721 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     6781 caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat
     6841 ctgtgcggta tttcacaccg catatatggt gcactctcag tacaatctgc tctgatgccg
     6901 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     6961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     7021 ggttttcacc gtcatcaccg aaacgcgcga
//
