LOCUS       sequence-431685-        3500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BxbI landing-pad mClover donor. Use with BxbI expressing plasmid to 
            swap payload into 4.721 landing-pad. Generates constitutively 
            expressing cell line..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3500)
  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 3500)
  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: pMIA10.5-Clover
FEATURES             Location/Qualifiers
     source          1..3500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    417..454
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      458..467
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(464..466,467..469,470..1180)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(509..530)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1117..1138
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1195..1232
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     primer_bind     complement(1279..1295)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1279..1295)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1292..1314)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1303..1319
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1327..1333,1334..1351,1352..1357))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1372..1393
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1510..1527)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1681..2269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1761..1780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2440..3231,3232..3300))
                     /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     3063..3082
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3301..3405)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3473..3491
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gagctagcta taggccggct
      421 tgtcgacgac ggcgctctcc gtcgtcagga tcatccggcc accatggtga gcaagggcga
      481 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
      541 caagttcagc gtccgcggcg agggcgaggg cgatgccacc aacggcaagc tgaccctgaa
      601 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcgg
      661 ctacggcgtg gcctgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
      721 gtccgccatg cccgaaggct acgtccagga gcgcaccatc tctttcaagg acgacggtac
      781 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
      841 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaactt
      901 caacagccac aacgtctata tcacggccga caagcagaag aacggcatca aggctaactt
      961 caagatccgc cacaacgttg aggacggcag cgtgcagctc gccgaccact accagcagaa
     1021 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gccatcagtc
     1081 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     1141 cgccgccggg attacacatg gcatggacga gctgtacaag taatagtacc ggccggcttg
     1201 tcgacgacgg cggtctccgt cgtcaggatc atccggttaa ttaaatccca atggcgcgcc
     1261 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     1321 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     1381 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     1441 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     1501 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     1561 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     1621 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     1681 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     1741 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     1801 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     1861 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     1921 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     1981 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2041 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     2101 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     2161 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2221 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2281 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2341 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2401 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     2461 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     2521 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     2581 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     2641 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     2701 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     2761 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     2821 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     2881 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     2941 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3001 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3061 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     3121 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     3181 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     3241 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     3301 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     3361 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     3421 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     3481 tatcacgagg ccctttcgtc
//
