LOCUS       sequence-431681-        6874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BxbI landing-pad HDR donor for targeting Pansio-1 GSH. CAGG-driven 
            Casp9-P2A-Bsd..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6874)
  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 6874)
  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-Pansio
FEATURES             Location/Qualifiers
     source          1..6874
                     /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        2354..2733
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2735..3012
                     /label=chicken beta-actin promoter
     intron          3013..4029
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3953..3975
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4037..4056
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    4216..4263
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             4287..4607
                     /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(4959..4970)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             5484..5510
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5526..5582
                     /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             5583..5981
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    5993..6040
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     polyA_signal    6073..6154
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     6098..6117
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(6478..6495)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6478..6494)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6487..6509)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6705..6724)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6824..6846
                     /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 ccaacagttg gccagttgca
     2041 aaaagagaat ctgcttgcat ggagtatgta aaaaagtcat attcttggaa agtagaatgg
     2101 acattgccag gggaggaagt gggaagttca atggatgcag agttttagtt ttgcaagatg
     2161 aaaagttcct agggatctat tgcacaacaa tgtgcatgta gttgacacta ctgaactgtg
     2221 tattgaaaaa gggttaagat tgcaaaaatc aatcttaggc aatttgattt ttcaagacac
     2281 agacatctac aaatgttatt attgaagaaa ccaatgcatc attacggatt atgcctttgc
     2341 atcatcgtcg atcgacattg attattgact agttattaat agtaatcaat tacggggtca
     2401 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     2461 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     2521 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     2581 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     2641 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     2701 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
     2761 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta
     2821 ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg
     2881 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
     2941 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
     3001 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
     3061 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
     3121 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg
     3181 tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg
     3241 gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc
     3301 ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg
     3361 agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc
     3421 gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa
     3481 ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc
     3541 cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg
     3601 ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc
     3661 cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg
     3721 tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg
     3781 cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa
     3841 atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct
     3901 cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc
     3961 ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt
     4021 ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa
     4081 ttctcgagga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     4141 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttaa
     4201 ttcccgggga tccgtggttt gtctggtcaa ccaccgcgct ctcagtggtg tacggtacaa
     4261 acccacacgt ggccaccatg ctcgagggag tgcaggtgga gactatctcc ccaggagacg
     4321 ggcgcacctt ccccaagcgc ggccagacct gcgtggtgca ctacaccggg atgcttgaag
     4381 atggaaagaa agttgattcc tcccgggaca gaaacaagcc ctttaagttt atgctaggca
     4441 agcaggaggt gatccgaggc tgggaagaag gggttgccca gatgagtgtg ggtcagagag
     4501 ccaaactgac tatatctcca gattatgcct atggtgccac tgggcaccca ggcatcatcc
     4561 caccacatgc cactctcgtc ttcgatgtgg agcttctaaa actggaatct ggcggtggat
     4621 ccggagtcga cggatttggt gatgtcggtg ctcttgagag tttgagggga aatgcagatt
     4681 tggcttacat cctgagcatg gagccctgtg gccactgcct cattatcaac aatgtgaact
     4741 tctgccgtga gtccgggctc cgcacccgca ctggctccaa catcgactgt gagaagttgc
     4801 ggcgtcgctt ctcctcgctg catttcatgg tggaggtgaa gggcgacctg actgccaaga
     4861 aaatggtgct ggctttgctg gagctggcgc ggcaggacca cggtgctctg gactgctgcg
     4921 tggtggtcat tctctctcac ggctgtcagg ccagccacct gcagttccca ggggctgtct
     4981 acggcacaga tggatgccct gtgtcggtcg agaagattgt gaacatcttc aatgggacca
     5041 gctgccccag cctgggaggg aagcccaagc tctttttcat ccaggcctgt ggtggggagc
     5101 agaaagacca tgggtttgag gtggcctcca cttcccctga agacgagtcc cctggcagta
     5161 accccgagcc agatgccacc ccgttccagg aaggtttgag gaccttcgac cagctggacg
     5221 ccatatctag tttgcccaca cccagtgaca tctttgtgtc ctactctact ttcccaggtt
     5281 ttgtttcctg gagggacccc aagagtggct cctggtacgt tgagaccctg gacgacatct
     5341 ttgagcagtg ggctcactct gaagacctgc agtccctcct gcttagggtc gctaatgctg
     5401 tttcggtgaa agggatttat aaacagatgc ctggttgctt taatttcctc cggaaaaaac
     5461 ttttctttaa aacatcagtc gactatccgt acgacgtacc agactacgca ctcgacggaa
     5521 gcggagccac taacttctcc ctgttgaaac aagcagggga tgtcgaagag aatcccgggc
     5581 caatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa
     5641 tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg
     5701 gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg
     5761 tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg
     5821 gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc
     5881 tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga
     5941 ttcgtgaatt gctgccctct ggttatgtgt gggagggcta ataaggtacc gtggtttgtc
     6001 tggtcaacca ccgcggtctc agtggtgtac ggtacaaacc caaccggtgt ttggacaaac
     6061 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     6121 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     6181 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     6241 tggtaacctg cagtagtgtc tccaggtgca aggaataaca gaaataaaag tcacatgatg
     6301 tgtgaacccc agagctttgg ggagactcct ataccaggtc tgaagtgacc taataggaag
     6361 tagctgttat tcatagtcaa tgccatatta atgaagagaa tgaacaacct caaaatggtc
     6421 taatatcatg actttatcta actgtatcta aatacactgc aggcatgcaa gcttggcact
     6481 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     6541 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     6601 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     6661 gcatctgtgc ggtatttcac accgcatata tggtgcactc tcagtacaat ctgctctgat
     6721 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     6781 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     6841 cagaggtttt caccgtcatc accgaaacgc gcga
//
