LOCUS       sequence-431690-        4616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BxbI landing-pad mClover-IRES-puro 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 4616)
  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 4616)
  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: pMIA10.53-Clover
FEATURES             Location/Qualifiers
     source          1..4616
                     /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      465..474
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(471..473,474..476,477..1187)
                     /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(516..537)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1124..1145
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1247..1821,1822..1824,1825..1833)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1414..1431)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1641..1660
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1834..2433
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1834..1853)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    2441..2478
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     primer_bind     complement(2626..2643)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2797..3385)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2877..2896)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3556..4347,4348..4416))
                     /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     4179..4198
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4417..4521)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4589..4607
                     /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 tcatccggac cggtgccacc atggtgagca
      481 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
      541 acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac ggcaagctga
      601 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
      661 ccttcggcta cggcgtggcc tgcttcagcc gctaccccga ccacatgaag cagcacgact
      721 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctct ttcaaggacg
      781 acggtaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
      841 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
      901 acaacttcaa cagccacaac gtctatatca cggccgacaa gcagaagaac ggcatcaagg
      961 ctaacttcaa gatccgccac aacgttgagg acggcagcgt gcagctcgcc gaccactacc
     1021 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagcc
     1081 atcagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1141 tcgtgaccgc cgccgggatt acacatggca tggacgagct gtacaagtaa taactggcgt
     1201 aatagcgaag aggcccgcac cgatcgccct tcccaacagt gaattccccc tctccctccc
     1261 ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata
     1321 tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg
     1381 tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt
     1441 tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag
     1501 cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc
     1561 cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga
     1621 tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg
     1681 cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat
     1741 gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc
     1801 tttgaaaaac acgatgataa tatggccaca accatgaccg agtacaagcc cacggtgcgc
     1861 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     1921 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     1981 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     2041 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     2101 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     2161 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     2221 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     2281 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgcccca caacctcccc
     2341 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     2401 acctggtgca tgacccgcaa gcccggtgcc taataaggcc ggcttgtcga cgacggcggt
     2461 ctccgtcgtc aggatcatcc ggttaattaa ccgatgccct tgagagcctt caacccagtc
     2521 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     2581 atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc tcactgactc
     2641 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2701 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2761 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2821 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2881 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2941 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3001 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3061 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3121 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3181 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3241 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3301 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3361 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3421 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3481 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3541 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3601 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3661 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3721 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3781 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3841 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3901 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3961 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4021 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4081 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4141 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4201 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4261 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4321 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4381 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4441 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4501 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     4561 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
