LOCUS       sequence-363150-        8670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-specific donor plasmid for intergrating a transcription unit 
            for expression of membrane-localised miRFP703.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8670)
  AUTHORS   Blanch-Asensio A, Grandela C, Brandao KO, de Korte T, Mei H, 
            Ariyurek Y, Yiangou L, Mol MPH, van Meer BJ, Kloet SL, Mummery CL, 
            Davis RP
  TITLE     STRAIGHT-IN enables high-throughput targeting of large DNA payloads 
            in human pluripotent stem cells.
  JOURNAL   Cell Rep Methods. 2022 Sep 22;2(10):100300. doi: 
            10.1016/j.crmeth.2022.100300. eCollection 2022 Oct 24.
  PUBMED    36313798
REFERENCE   2  (bases 1 to 8670)
  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: "Cell Rep 
            Methods."; date: "2022-09-22"; volume: "2(10):100300. doi"; pages: "
            10.1016/j.crmeth.2022.100300. eCollection 2022 Oct 24"
COMMENT     Sequence Label: Bxb1_miRFP703
FEATURES             Location/Qualifiers
     source          1..8670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    86..110
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        186..1364
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          417..1355
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1312..1332
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1372..1381
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1384..1421
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    complement(1430..1463)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        1604..1907
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          2152..3168
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3092..3114
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3176..3195
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3230..3239
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3281..4228
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 703 nm (Shcherbakova et al., 2016)"
                     /label=miRFP703
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSLVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPIHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     polyA_signal    4350..4471
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4387..4406)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4441..4460
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(4532..4564)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(4572..4589)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4572..4588)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    4625..4649
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      4737..5447
                     /label=oriV
                     /note="incP origin of replication"
     promoter        5468..5572
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5573..6367
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      6538..7126
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7027..7046
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             7462..8610
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
ORIGIN
        1 ggacagtagt catggttgat gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc
       61 ggtcggggag ctgttggctg gctggtggca ggatatattg tggtgtaaac aaattgacgc
      121 ttagacaact taataacaca ttgcggacgt ttttaatgta ctggggttga acactctgtg
      181 ccattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
      241 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
      301 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
      361 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa
      421 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt
      481 gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg
      541 ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg
      601 cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg
      661 ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt
      721 tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg
      781 gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc
      841 tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg
      901 tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg
      961 caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat
     1021 ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct
     1081 ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc
     1141 tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg
     1201 cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga
     1261 tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc
     1321 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgaggaatt cgccaccatg
     1381 gccggcttgt cgacgacggc ggtctccgtc gtcaggatca tccggatcca taacttcgta
     1441 tagcatacat tatacgaagt tatcatgata ttcggcaagc aggcatcgta gctgccgatg
     1501 tatcacctct cgcaaggttt tgacgggttt actagaattc gagctcggag actaactacg
     1561 gtgaaagaga cttagaagga gattgagaga tactggagtt ccgcgttaca taacttacgg
     1621 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     1681 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     1741 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     1801 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1861 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
     1921 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
     1981 ttatttttta attattttgt gcagcgatgg gggcgggggg ggggsnnnnn gggcggggcg
     2041 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     2101 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     2161 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
     2221 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     2281 aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag
     2341 gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg
     2401 tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
     2461 ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc
     2521 ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg
     2581 gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc
     2641 cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg
     2701 ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct
     2761 cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga
     2821 ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt
     2881 cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc
     2941 gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt
     3001 gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac
     3061 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc
     3121 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
     3181 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tccgatcgcg ccaccatggg
     3241 ctgcgtgtgc agcagcaacc ccgagctacc ggtcgccaca atggtagcag gtcatgcctc
     3301 tggcagcccc gcattcggga ccgcctctca ttcgaattgc gaacatgaag agatccacct
     3361 cgccggctcg atccagccgc atggcgcgct tctggtcgtc agcgaacatg atcatcgcgt
     3421 catccaggcc agcgccaacg ccgcggaatt tctgaatctc ggaagcgtac tcggcgttcc
     3481 gctcgccgag atcgacggcg atctgttgat caagatcctg ccgcatctcg atcccaccgc
     3541 cgaaggcatg ccggtcgcgg tgcgctgccg gatcggcaat ccctctacgg agtactgcgg
     3601 tctgatgcat cggcctccgg aaggcgggct gatcatcgaa ctcgaacgtg ccggcccgtc
     3661 gatcgatctg tcaggcacgc tggcgccggc gctggagcgg atccgcacgg cgggttcact
     3721 gcgcgcgctg tgcgatgaca ccgtgctgct gtttcagcag tgcaccggct acgaccgggt
     3781 gatggtgtat cgtttcgatg agcaaggcca cggcctggta ttctccgagt gccatgtgcc
     3841 tgggctcgaa tcctatttcg gcaaccgcta tccgtcgtcg ctggtcccgc agatggcgcg
     3901 gcagctgtac gtgcggcagc gcgtccgcgt gctggtcgac gtcacctatc agccggtgcc
     3961 gctggagccg cggctgtcgc cgctgaccgg gcgcgatctc gacatgtcgg gctgcttcct
     4021 gcgctcgatg tcgccgatcc atctgcagtt cctgaaggac atgggcgtgc gcgccaccct
     4081 ggcggtgtcg ctggtggtcg gcggcaagct gtggggcctg gttgtctgtc accattatct
     4141 gccgcgcttc atccgtttcg agctgcgggc gatctgcaaa cggctcgccg aaaggatcgc
     4201 gacgcggatc accgcgcttg agagctaagc ggccgcgact ctagatcata atcagccata
     4261 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     4321 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     4381 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4441 gtggtttgtc caaactcatc aatgtatctt aaggcgcttt acgaggatgc acatgtgacc
     4501 gagggaggag ctgcaagaac tcttcctcac gataacttcg tataggagac tttatacgaa
     4561 gttaagcgct cactggccgt cgttttacag ggacacgaag tgatccgttt aaactatcag
     4621 tgtttgacag gatatattgg cgggtaaacc taagagaaaa gagcgtttat tagaataatc
     4681 ggatatttaa aagggcgtga aaaggtttat ccgttcgtcc atttgtatgt gccagccgcc
     4741 tttgcgacgc tcaccgggct ggttgccctc gccgctgggc tggcggccgt ctatggccct
     4801 gcaaacgcgc cagaaacgcc gtcgaagccg tgtgcgagac accgcggccg ccggcgttgt
     4861 ggatacctcg cggaaaactt ggccctcact gacagatgag gggcggacgt tgacacttga
     4921 ggggccgact cacccggcgc ggcgttgaca gatgaggggc aggctcgatt tcggccggcg
     4981 acgtggagct ggccagcctc gcaaatcggc gaaaacgcct gattttacgc gagtttccca
     5041 cagatgatgt ggacaagcct ggggataagt gccctgcggt attgacactt gaggggcgcg
     5101 actactgaca gatgaggggc gcgatccttg acacttgagg ggcagagtgc tgacagatga
     5161 ggggcgcacc tattgacatt tgaggggctg tccacaggca gaaaatccag catttgcaag
     5221 ggtttccgcc cgtttttcgg ccaccgctaa cctgtctttt aacctgcttt taaaccaata
     5281 tttataaacc ttgtttttaa ccagggctgc gccctgtgcg cgtgaccgcg cacgccgaag
     5341 gggggtgccc ccccttctcg aaccctcccg gcccgctaac gcgggcctcc catcccccca
     5401 ggggctgcgc ccctcggccg cgaacggcct caccccaaaa atggcagcgc tggccaattc
     5461 ccgagtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5521 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatggctaa
     5581 aatgagaata tcaccggaat tgaaaaaact gatcgaaaaa taccgctgcg taaaagatac
     5641 ggaaggaatg tctcctgcta aggtatataa gctggtggga gaaaatgaaa acctatattt
     5701 aaaaatgacg gacagccggt ataaagggac cacctatgat gtggaacggg aaaaggacat
     5761 gatgctatgg ctggaaggaa agctgcctgt tccaaaggtc ctgcactttg aacggcatga
     5821 tggctggagc aatctgctca tgagtgaggc cgatggcgtc ctttgctcgg aagagtatga
     5881 agatgaacaa agccctgaaa agattatcga gctgtatgcg gagtgcatca ggctctttca
     5941 ctccatcgac atatcggatt gtccctatac gaatagctta gacagccgct tagccgaatt
     6001 ggattactta ctgaataacg atctggccga tgtggattgc gaaaactggg aagaggacac
     6061 tccatttaaa gatccgcgcg agctgtatga ttttttaaag acggaaaagc ccgaagagga
     6121 acttgtcttt tcccacggcg acctgggaga cagcaacatc tttgtgaaag atggcaaagt
     6181 aagtggcttt attgatcttg ggagaagcgg cagggcggac aagtggtatg acattgcctt
     6241 ctgcgtccgg tcgctcaggg aggatatcgg ggaagaacag tatgtcgagc tattttttga
     6301 cttactgggg atcaagcctg attgggagaa aataaaatat tatattttac tggatgaatt
     6361 gttttagctg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6421 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6481 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6541 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6601 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6661 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6721 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6781 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6841 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6901 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6961 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7021 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7081 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7141 ggccttttta cggttcctgg cagatcctag atgtggcgca acgatgccgg cgacaagcag
     7201 gagcgcaccg acttcttccg catcaagtgt tttggctctc aggccgaggc ccacggcaag
     7261 tatttgggca aggggtcgct ggtattcgtg cagggcaaga ttcggaatac caagtacgag
     7321 aaggacggcc agacggtcta cgggaccgac ttcattgccg ataaggtgga ttatctggac
     7381 accaaggcac caggcgggtc aaatcaggaa taagggcaca ttgccccggc gtgagtcggg
     7441 gcaatcccgc aaggagggtg aatgaatcgg acgtttgacc ggaaggcata caggcaagaa
     7501 ctgatcgacg cggggttttc cgccgaggat gccgaaacca tcgcaagccg caccgtcatg
     7561 cgtgcgcccc gcgaaacctt ccagtccgtc ggctcgatgg tccagcaagc tacggccaag
     7621 atcgagcgcg acagcgtgca actggctccc cctgccctgc ccgcgccatc ggccgccgtg
     7681 gagcgttcgc gtcgtcttga acaggaggcg gcaggtttgg cgaagtcgat gaccatcgac
     7741 acgcgaggaa ctatgacgac caagaagcga aaaaccgccg gcgaggacct ggcaaaacag
     7801 gtcagcgagg ccaagcaggc cgcgttgctg aaacacacga agcagcagat caaggaaatg
     7861 cagctttcct tgttcgatat tgcgccgtgg ccggacacga tgcgagcgat gccaaacgac
     7921 acggcccgct ctgccctgtt caccacgcgc aacaagaaaa tcccgcgcga ggcgctgcaa
     7981 aacaaggtca ttttccacgt caacaaggac gtgaagatca cctacaccgg cgtcgagctg
     8041 cgggccgacg atgacgaact ggtgtggcag caggtgttgg agtacgcgaa gcgcacccct
     8101 atcggcgagc cgatcacctt cacgttctac gagctttgcc aggacctggg ctggtcgatc
     8161 aatggccggt attacacgaa ggccgaggaa tgcctgtcgc gcctacaggc gacggcgatg
     8221 ggcttcacgt ccgaccgcgt tgggcacctg gaatcggtgt cgctgctgca ccgcttccgc
     8281 gtcctggacc gtggcaagaa aacgtcccgt tgccaggtcc tgatcgacga ggaaatcgtc
     8341 gtgctgtttg ctggcgacca ctacacgaaa ttcatatggg agaagtaccg caagctgtcg
     8401 ccgacggccc gacggatgtt cgactatttc agctcgcacc gggagccgta cccgctcaag
     8461 ctggaaacct tccgcctcat gtgcggatcg gattccaccc gcgtgaagaa gtggcgcgag
     8521 caggtcggcg aagcctgcga agagttgcga ggcagcggcc tggtggaaca cgcctgggtc
     8581 aatgatgacc tggtgcattg caaacgctag ggccttgtgg ggtcagttcc ggctgggggt
     8641 tcagcagccc ctgctcggat ctgttggacc
//
