LOCUS       sequence-437331-        5801 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expressing evolved 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: evoBxb1
FEATURES             Location/Qualifiers
     source          1..5801
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        complement(834..938)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1006..1024
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1051..1056,1057..1074,1075..1081)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    1089..1105
                     /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)."
     primer_bind     1094..1116
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     enhancer        1135..1438
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1439..1642
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1593..1613
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1639..1663
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1758..1776
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1758..1775
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1814..3316
                     /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
                     REPQGREWSATALKRSMISEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAE
                     LVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTV
                     AMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNAELVDLTSLIGSPAYRAG
                     SPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSM
                     NVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS"
     polyA_signal    3728..3862
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3765..3784)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3819..3838
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3988..4442
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4075..4094)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4284..4305
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             join(4729..4797,4798..5589)
                     /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     complement(4947..4966)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    5691..5724
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagagctt gcaattcgcg
      841 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
      901 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
      961 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     1021 aggccctttc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat
     1081 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agcatcgtgc aggtcgttac
     1141 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     1201 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     1261 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     1321 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     1381 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1441 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     1501 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     1561 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     1621 ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga ggcgccatcc
     1681 acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga ctctagccta
     1741 ggcttttgca aaaagctatt taggtgacac tatagaaggt acgcctgcag gtaccggtcc
     1801 ggaattcgcc cttatgagag ccctggtagt catccgcctg tcccgcgtca ccgatgctac
     1861 gacttcaccg gagcgtcagc tggagtcttg ccagcagctc tgcgcccagc gcggctggga
     1921 cgtcgtcggg gtagcggagg atctggacgt ctccggggcg gtcgatccgt tcgaccggaa
     1981 gcgcagaccg aacctggccc ggtggctagc gttcgaggag caaccgttcg acgcgatcgt
     2041 ggcgtaccgg gtagaccggt tgacccgatc gatccggcat ctgcagcagc tggtccactg
     2101 ggccgaggac cacaagaagc tggtcgtctc cgcgaccgaa gcgcacttcg atacgacgac
     2161 gccgtttgcg gcggtcgtca tcgcgcttat gggaacggtg gcgcagatgg aattagaagc
     2221 gatcaaagag cggaaccgtt cggctgcgca tttcaatatc cgcgccggga aataccgagg
     2281 atccctgccg ccgtggggat acctgcctac gcgcgtggac ggggagtggc ggctggtgcc
     2341 ggaccctgtg cagcgagagc gcatcctcga ggtgtatcac cgcgtcgtcg acaaccacga
     2401 gccgctgcac ctggtggccc acgacctgaa ccggcgtggt gtcctgtcgc cgaaggacta
     2461 cttcgcgcag ctgcaaggcc gcgagccgca gggccgggag tggtcggcta ccgcgctgaa
     2521 gcgatcgatg atctccgagg cgatgctcgg gtacgcgact ctgaacggta agaccgtccg
     2581 agacgacgac ggagccccgc tggtgcgggc tgagccgatc ctgacccgtg agcagctgga
     2641 ggcgctgcgc gccgagctcg tgaagacctc ccgggcgaag cccgcggtgt ctaccccgtc
     2701 gctgctgctg cgggtgttgt tctgtgcggt gtgcggggag cccgcgtaca agttcgccgg
     2761 gggaggacgt aagcacccgc gctaccgctg ccgctcgatg gggttcccga agcactgcgg
     2821 gaacggcacg gtggcgatgg ccgagtggga cgcgttctgc gaggagcagg tgctggatct
     2881 gctcggggac gcggagcgtc tggagaaagt ctgggtagcc ggctcggact ccgcggtcga
     2941 actcgcggag gtgaacgcgg agctggtgga cctgacgtcg ctgatcggct ccccggccta
     3001 ccgggccggc tctccgcagc gagaagcact ggatgcccgt attgcggcgc tggccgcgcg
     3061 gcaagaggag ctggagggtc tagaggctcg cccgtctggc tgggagtggc gcgagaccgg
     3121 gcagcggttc ggggactggt ggcgggagca ggacaccgcg gcaaagaaca cctggcttcg
     3181 gtcgatgaac gttcggctga cgttcgacgt ccgcggtggg ctgactcgca cgatcgactt
     3241 cggggatctg caggagtacg agcagcatct caggctcggc agcgtggtcg aacggctaca
     3301 caccgggatg tcgtagaagg gcgaattccc gggtcgacga gctcactagc ttgggatctt
     3361 tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta cagagattta
     3421 aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactag ctgcatatgc
     3481 ttgctgcttg agagttttgc ttactgagta tgatttatga aaatattata cacaggagct
     3541 agtgattcta attgtttgtg tattttagat tcacagtccc aaggctcatt tcaggcccct
     3601 cagtcctcac agtctgttca tgatcataat cagccatacc acatttgtag aggttttact
     3661 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     3721 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     3781 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     3841 tgtatcttat catgtctgga tcgatcctgc attaatgaat cggccaacgc gcggggagag
     3901 gcggtttgcg tattggctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     3961 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     4021 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     4081 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4141 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4201 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4261 cgttggagtc cacgttctta atagtggact cttgttccaa actggaacaa cactcaaccc
     4321 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4381 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     4441 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4501 cattcaaata tgtatccgct catgccaggt cttggactgg tgagaacggc ttgctcggca
     4561 gcttcgatgt gtgctggagg gagaataaag gtctaagatg tgcgatagag ggaagtcgca
     4621 ttgaattatg tgctgtgtag ggatcgctgg tatcaaatat gtgtgcccac ccctggcatg
     4681 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4741 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4801 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4861 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4921 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4981 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5041 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5101 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5161 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5221 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5281 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5341 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5401 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5461 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5521 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5581 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5641 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgcc ataacttcgt
     5701 ataatgtatg ctatacgagg ttatggcatg accaaaatcc cttaacgtga gttttcgttc
     5761 cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
