LOCUS       sequence-431689-        5500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BxbI landing-pad Tet-On-T2A-puro-pA pA-GFP-TRE3G donor. Use with 
            BxbI expressing plasmid to swap payload into 4.721 landing-pad. 
            Generates inducible cell line..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5500)
  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 5500)
  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: pMIA10.7
FEATURES             Location/Qualifiers
     source          1..5500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     202..224
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     216..233
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     217..233
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    255..292
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             302..1045
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             1055..1108
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(1109..1128)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1605..1625
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1715..1734)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1780..1835
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     polyA_signal    1954..2035
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1991..2010)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2045..2064
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(2078..2743)
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     CDS             complement(2078..2743)
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     promoter        complement(2756..3134)
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     primer_bind     complement(2823..2847)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    2892..2910
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2928..2946
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2964..2982
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3000..3018
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3036..3054
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3072..3090
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3108..3126
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3149..3186
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     primer_bind     complement(3233..3249)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3233..3249)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3246..3268)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3257..3273
                     /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(3281..3287,3288..3305,3306..3311))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3326..3347
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3519..4107)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3599..3618)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4278..5069,5070..5138))
                     /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     4901..4920
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5139..5243)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5311..5329
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5367..5389)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     join(5489..5500,1..8)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
       61 aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
      121 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
      181 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattga
      241 gctagctata ggccggcttg tcgacgacgg cgctctccgt cgtcaggatc atccggccac
      301 catgtctaga ctggacaaga gcaaagtcat aaactctgct ctggaattac tcaatggagt
      361 cggtatcgaa ggcctgacga caaggaaact cgctcaaaag ctgggagttg agcagcctac
      421 cctgtactgg cacgtgaaga acaagcgggc cctgctcgat gccctgccaa tcgagatgct
      481 ggacaggcat catacccact cctgccccct ggaaggcgag tcatggcaag actttctgcg
      541 gaacaacgcc aagtcatacc gctgtgctct cctctcacat cgcgacgggg ctaaagtgca
      601 tctcggcacc cgcccaacag agaaacagta cgaaaccctg gaaaatcagc tcgcgttcct
      661 gtgtcagcaa ggcttctccc tggagaacgc actgtacgct ctgtccgccg tgggccactt
      721 tacactgggc tgcgtattgg aggaacagga gcatcaagta gcaaaagagg aaagagagac
      781 acctaccacc gattctatgc ccccacttct gaaacaagca attgagctgt tcgaccggca
      841 gggagccgaa cctgccttcc ttttcggcct ggaactaatc atatgtggcc tggagaaaca
      901 gctaaagtgc gaaagcggcg ggccgaccga cgcccttgac gattttgact tagacatgct
      961 cccagccgat gcccttgacg actttgacct tgatatgctg cctgctgacg ctcttgacga
     1021 ttttgacctt gacatgctcc ccgggggaag cggagagggc agaggaagtc tgctaacatg
     1081 cggtgatgtc gaggagaatc ctggcccaat gaccgagtac aagcccacgg tccgcctggc
     1141 tacgcgcgac gacgtcccta gggccgtccg caccctcgcc gccgcattcg ccgactaccc
     1201 cgccacgcgc cacaccgtcg atccggatcg ccacatcgag cgggtcaccg aactgcagga
     1261 actgttcctc acccgcgtgg ggctcgacat cggaaaggtg tgggtcgcgg acgacggcgc
     1321 cgccgtggcg gtctggacca ccccggagag cgtcgaagct ggtgcagtgt tcgccgagat
     1381 cggccctcgt atggccgaat tgagcggttc tcgtctggcc gcgcagcaac agatggaagg
     1441 cctgctcgcg ccgcaccgtc ccaaggagcc cgcgtggttc ctggctaccg tcggagtctc
     1501 ccccgatcac cagggaaagg gactgggcag cgccgtcgtg ctccccggag tggaagcggc
     1561 tgaacgtgca ggggtgcccg ccttcctgga gacctccgct ccccgcaacc tccccttcta
     1621 cgagcggctc ggcttcaccg tcaccgccga tgtcgaggtc cctgaaggac cgcgcacctg
     1681 gtgtatgaca cgcaagcccg gtgcttgata aggatctttt tccctctgcc aaaaattatg
     1741 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca
     1801 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aagcaaaccg gtccctctta
     1861 agtaccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     1921 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     1981 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     2041 agttgtggtt tgtccaaact catcgagctc gagatctggc gaaggcgatg ggggtcttga
     2101 aggcgtgctg gtactccacg atgcccagct cggtgttgct gtgcagctcc tccacgcggc
     2161 ggaaggcgaa catggggccc ccgttctgca ggatgctggg gtggatggcg ctcttgaagt
     2221 gcatgtggct gtccaccacg aagctgtagt agccgccgtc gcgcaggctg aaggtgcggg
     2281 cgaagctgcc caccagcacg ttatcgccca tggggtgcag gtgctccacg gtggcgttgc
     2341 tgcggatgat cttgtcggtg aagatcacgc tgtcctcggg gaagccggtg cccaccacct
     2401 tgaagtcgcc gatcacgcgg ccggcctcgt agcggtagct gaagctcacg tgcagcacgc
     2461 cgccgtcctc gtacttctcg atgcgggtgt tggtgtagcc gccgttgttg atggcgtgca
     2521 ggaaggggtt ctcgtagccg ctggggtagg tgccgaagtg gtagaagccg tagcccatca
     2581 cgtggctcag caggtagggg ctgaaggtca gggcgccttt ggtgctcttc atcttgttgg
     2641 tcatgcggcc ctgctcgggg gtgccctctc cgccgcccac cagctcgaac tccacgccgt
     2701 tcagggtgcc ggtgatgcgg cactcgatct tcatggcggg catggtggcg gccgctttac
     2761 gagggtagga agtggtacgg aaagttggta taagacaaaa gtgttgtgga attgctccag
     2821 gcgatctgac ggttcactaa acgagctctg cttttatagg cgcccaccgt acacgcctaa
     2881 agcttatacg ttctctatca ctgataggga gtaaactgga tatacgttct ctatcactga
     2941 tagggagtaa actgtagata cgttctctat cactgatagg gagtaaactg gtcatacgtt
     3001 ctctatcact gatagggagt aaactcctta tacgttctct atcactgata gggagtaaag
     3061 tctgcatacg ttctctatca ctgataggga gtaaactctt catacgttct ctatcactga
     3121 tagggagtaa actcgaggag ctaaggccgg cttgtcgacg acggcggtct ccgtcgtcag
     3181 gatcatccgg ttaattaaat cccaatggcg cgccgagctt ggcgtaatca tggtcatagc
     3241 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3301 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3361 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3421 gcgcggggag aggcggtttg cgtattgggc gctcttccgc tttgagcaaa aggccagcaa
     3481 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3541 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3601 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3661 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3721 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3781 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3841 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3901 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     3961 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4021 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4081 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4141 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4201 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4261 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4321 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4381 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4441 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4501 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4561 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4621 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4681 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4741 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4801 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4861 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4921 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4981 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5041 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5101 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5161 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5221 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     5281 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     5341 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     5401 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     5461 ggcgggtgtc ggggctggct taactatgcg gcatcagagc
//
