LOCUS       sequence-473210-        5801 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expressing evolved and engineered Bxb1 in the mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5801)
  AUTHORS   Pandey S, Gao XD, Krasnow NA, McElroy A, Tao YA, Duby JE, Steinbeck 
            BJ, McCreary J, Pierce SE, Tolar J, Meissner TB, Chaikof EL, Osborn 
            MJ, Liu DR
  TITLE     Efficient site-specific integration of large genes in mammalian 
            cells via continuously evolved recombinases and prime editing.
  JOURNAL   Nat Biomed Eng. 2024 Jun 10. doi: 10.1038/s41551-024-01227-1.
  PUBMED    38858586
REFERENCE   2  (bases 1 to 5801)
  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: "Nat Biomed 
            Eng. 2024 Jun 10. doi: 10.1038/s41551-024-01227-1."
COMMENT     Sequence Label: eeBxb1
FEATURES             Location/Qualifiers
     source          1..5801
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1051..1069)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1052..1069)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1164..1188)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(1185..1388)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1214..1234)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1389..1692
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(1711..1733)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1722..1738
                     /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(1746..1752,1753..1770,1771..1776))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(1803..1821)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1889..1993
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2067..2084)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2238..2826)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2318..2337)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(2904..2937)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(3039..3830,3831..3899))
                     /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     3662..3681
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4186..4640)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4323..4344)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4534..4553
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    4766..4900
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4790..4809)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4844..4863
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(5312..5801,1..1013))
                     /codon_start=1
                     /transl_table=11
                     /gene="Bxb1"
                     /product="Bxb1 integrase"
                     /label=Bxb1 integrase
                     /note="Bxb1 large serine integrase from Mycobacterium 
                     bacteriophage Bxb1 (Ghosh et al. 2003)."
                     /protein_id="AFF61396.1"
                     /translation="MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLD
                     VSGAVDPFDRKRRPNLARWLAFEEQPFDAIVAYRVDRLTRSIRHLQQLVHWAEDHKKLV
                     VSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGY
                     LPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQG
                     REPQGRKWSATALKRSMISEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAE
                     LVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTV
                     AMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAIELAEVNAELVDLTSLIGSPAYRAG
                     SPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSM
                     NVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS"
ORIGIN
        1 ccgttcccgc agtgcttcgg gaaccccatc gagcggcagc ggtagcgcgg gtgcttacgt
       61 cctcccccgg cgaacttgta cgcgggctcc ccgcacaccg cgcagaacaa cacccgcagc
      121 agcagcgacg gggtagacac cgcgggcttc gcccgggagg tcttcacgag ctcggcgcgc
      181 agcgcctcca gctgctcacg ggtcaggatc ggctcagccc gcaccagcgg ggctccgtcg
      241 tcgtctcgga cggtcttacc gttcagagtc gcgtacccga gcatcgcctc ggagatcatc
      301 gatcgcttca gcgcggtagc cgaccacttc cggccctgcg gctcgcggcc ttgcagctgc
      361 gcgaagtagt ccttcggcga caggacacca cgccggttca ggtcgtgggc caccaggtgc
      421 agcggctcgt ggttgtcgac gacgcggtga tacacctcga ggatgcgctc tcgctgcaca
      481 gggtccggca ccagccgcca ctccccgtcc acgcgcgtag gcaggtatcc ccacggcggc
      541 agggatcctc ggtatttccc ggcgcggata ttgaaatgcg cagccgaacg gttccgctct
      601 ttgatcgctt ctaattccat ctgcgccacc gttcccataa gcgcgatgac gaccgccgca
      661 aacggcgtcg tcgtatcgaa gtgcgcttcg gtcgcggaga cgaccagctt cttgtggtcc
      721 tcggcccagt ggaccagctg ctgcagatgc cggatcgatc gggtcaaccg gtctacccgg
      781 tacgccacga tggcgtcgaa cggttgctcc tcgaacgcta gccaccgggc caggttcggt
      841 ctgcgcttcc ggtcgaacgg atcgaccgcc ccggagacgt ccagatcctc cgctaccccg
      901 acgacgtccc agccgcgctg ggcgcagagc tgctggcaag actccagctg acgctccggt
      961 gaagtcgtag catcggtgac gcgggacagg cggatgacta ccagggctct cataagggcg
     1021 aattccggac cggtacctgc aggcgtacct tctatagtgt cacctaaata gctttttgca
     1081 aaagcctagg ctagagtccg gaggctggat cggtcccggt gtcttctatg gaggtcaaaa
     1141 cagcgtggat ggcgcctcca ggcgatctga cggttcacta aacgagctct gcttatatag
     1201 acctcccacc gtacacgcct accgcccatt tgcgtcaatg gggcggagtt gttacgacat
     1261 tttggaaagt cccgttgatt ttggtgccaa aacaaactcc cattgacgtc aatggggtgg
     1321 agacttggaa atccccgtga gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac
     1381 cgcatcacca tggtaatagc gatgactaat acgtagatgt actgccaagt aggaaagtcc
     1441 cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt gacgtcaata
     1501 gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt ttaccgtaaa
     1561 tactccaccc attgacgtca atggaaagtc cctattggcg ttactatggg aacatacgtc
     1621 attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc atttaccgta
     1681 agttatgtaa cgacctgcac gatgctgttt cctgtgtgaa attgttatcc gctcacaatt
     1741 ccacacatta tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgaaa
     1801 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     1861 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1921 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1981 aaaaagcgcg aattgcaagc tctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2041 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2101 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2161 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2221 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2281 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2341 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2401 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2461 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2521 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2581 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2641 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2701 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2761 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2821 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2881 cgttaaggga ttttggtcat gccataacct cgtatagcat acattatacg aagttatggc
     2941 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3001 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3061 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3121 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3181 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3241 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3301 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3361 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3421 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3481 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3541 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3601 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3661 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3721 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3781 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3841 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3901 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gccaggggtg
     3961 ggcacacata tttgatacca gcgatcccta cacagcacat aattcaatgc gacttccctc
     4021 tatcgcacat cttagacctt tattctccct ccagcacaca tcgaagctgc cgagcaagcc
     4081 gttctcacca gtccaagacc tggcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4141 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     4201 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     4261 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     4321 ttccagtttg gaacaagagt ccactattaa gaacgtggac tccaacgtca aagggcgaaa
     4381 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     4441 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     4501 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     4561 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     4621 tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg
     4681 atcggtgcgg gcctcttcgc tattacgcca gccaatacgc aaaccgcctc tccccgcgcg
     4741 ttggccgatt cattaatgca ggatcgatcc agacatgata agatacattg atgagtttgg
     4801 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     4861 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     4921 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     4981 caaatgtggt atggctgatt atgatcatga acagactgtg aggactgagg ggcctgaaat
     5041 gagccttggg actgtgaatc taaaatacac aaacaattag aatcactagc tcctgtgtat
     5101 aatattttca taaatcatac tcagtaagca aaactctcaa gcagcaagca tatgcagcta
     5161 gtttaacaca ttatacactt aaaaatttta tatttacctt agagctttaa atctctgtag
     5221 gtagtttgtc caattatgtc acaccacaga agtaaggttc cttcacaaag atcccaagct
     5281 agtgagctcg tcgacccggg aattcgccct tctacgacat cccggtgtgt agccgttcga
     5341 ccacgctgcc gagcctgaga tgctgctcgt actcctgcag atccccgaag tcgatcgtgc
     5401 gagtcagccc gccgcggacg tcgaacgtca gccgaacgtt catcgaccga agccaggtgt
     5461 tctttgccgc ggtgtcctgc tcccgccacc agtccccgaa ccgctgcccg gtctcgcgcc
     5521 actcccagcc agacgggcga gcctctagac cctccagctc ctcttgccgc gcggccagcg
     5581 ccgcaatacg ggcatccagt gcttctcgct gcggagagcc ggcccggtag gccggggagc
     5641 cgatcagcga cgtcaggtcc accagctccg cgttcacctc cgcgagttcg atcgcggagt
     5701 ccgagccggc tacccagact ttctccagac gctccgcgtc cccgagcaga tccagcacct
     5761 gctcctcgca gaacgcgtcc cactcggcca tcgccaccgt g
//
