LOCUS       sequence-440249-        4984 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for evolved and engineered Bxb1 mRNA in vitro transcription 
            template.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4984)
  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 4984)
  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_IVT
FEATURES             Location/Qualifiers
     source          1..4984
                     /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"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1062..1286
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(1394..2896)
                     /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"
     promoter        complement(2996..3199)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3025..3045)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3200..3579
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3751..3770
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3849..3953
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3954..4022,4023..4814)
                     /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(4172..4191)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
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 tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccctag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtatc gagccccagc tggttctttc cgcctcagaa gccatagagc ccaccgcatc
     1081 cccagcatgc ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc
     1141 ccccagaata gaatgacacc tactcagaca atgcgatgca attttctcat tttattagga
     1201 aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac
     1261 agatggctgg caactagaag gcacagtcgc ttcctactca ggctttattc aaagaccaag
     1321 aggtacaggt gcaagggaga gaagaagggc atggccagaa ggcaagcccc gcagaaggca
     1381 gcttaattaa ttactacgac atcccggtgt gtagccgttc gaccacgctg ccgagcctga
     1441 gatgctgctc gtactcctgc agatccccga agtcgatcgt gcgagtcagc ccgccgcgga
     1501 cgtcgaacgt cagccgaacg ttcatcgacc gaagccaggt gttctttgcc gcggtgtcct
     1561 gctcccgcca ccagtccccg aaccgctgcc cggtctcgcg ccactcccag ccagacgggc
     1621 gagcctctag accctccagc tcctcttgcc gcgcggccag cgccgcaata cgggcatcca
     1681 gtgcttctcg ctgcggagag ccggcccggt aggccgggga gccgatcagc gacgtcaggt
     1741 ccaccagctc cgcgttcacc tccgcgagtt cgatcgcgga gtccgagccg gctacccaga
     1801 ctttctccag acgctccgcg tccccgagca gatccagcac ctgctcctcg cagaacgcgt
     1861 cccactcggc catcgccacc gtgccgttcc cgcagtgctt cgggaacccc atcgagcggc
     1921 agcggtagcg cgggtgctta cgtcctcccc cggcgaactt gtacgcgggc tccccgcaca
     1981 ccgcgcagaa caacacccgc agcagcagcg acggggtaga caccgcgggc ttcgcccggg
     2041 aggtcttcac gagctcggcg cgcagcgcct ccagctgctc acgggtcagg atcggctcag
     2101 cccgcaccag cggggctccg tcgtcgtctc ggacggtctt accgttcaga gtcgcgtacc
     2161 cgagcatcgc ctcggagatc atcgatcgct tcagcgcggt agccgaccac ttccggccct
     2221 gcggctcgcg gccttgcagc tgcgcgaagt agtccttcgg cgacaggaca ccacgccggt
     2281 tcaggtcgtg ggccaccagg tgcagcggct cgtggttgtc gacgacgcgg tgatacacct
     2341 cgaggatgcg ctctcgctgc acagggtccg gcaccagccg ccactccccg tccacgcgcg
     2401 taggcaggta tccccacggc ggcagggatc ctcggtattt cccggcgcgg atattgaaat
     2461 gcgcagccga acggttccgc tctttgatcg cttctaattc catctgcgcc accgttccca
     2521 taagcgcgat gacgaccgcc gcaaacggcg tcgtcgtatc gaagtgcgct tcggtcgcgg
     2581 agacgaccag cttcttgtgg tcctcggccc agtggaccag ctgctgcaga tgccggatcg
     2641 atcgggtcaa ccggtctacc cggtacgcca cgatggcgtc gaacggttgc tcctcgaacg
     2701 ctagccaccg ggccaggttc ggtctgcgct tccggtcgaa cggatcgacc gccccggaga
     2761 cgtccagatc ctccgctacc ccgacgacgt cccagccgcg ctgggcgcag agctgctggc
     2821 aagactccag ctgacgctcc ggtgaagtcg tagcatcggt gacgcgggac aggcggatga
     2881 ctaccagggc tctcatggtg gctcttatat ttcttcttac tcttcttttc tctcttattt
     2941 ccttatagtg tgtcgtatta ggtaccgagc tcgagatctg acggttcact aaaccagctc
     3001 tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt
     3061 tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt
     3121 caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt
     3181 actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag
     3241 taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat
     3301 tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag
     3361 tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg
     3421 gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc
     3481 catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg
     3541 taattgatta ctattaataa ctagtcaata atcaatgtca acgcgtatat ctggcccgta
     3601 catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt cttcatgcaa ttgtcggtca
     3661 agccttgcct tgttgtagct taaattttgc tcgcgcacta ctcagcgacc tccaacacac
     3721 aagcagggag cagatactgg cttaactatg cggcatcaga gcagattgta ctgagagtag
     3781 accctagggg atcgggagat cgatctcccg atccgtcgac gtcaggtggc acttttcggg
     3841 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3901 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     3961 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4021 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4081 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4141 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4201 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4261 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4321 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4381 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4441 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     4501 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4561 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4621 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4681 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4741 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4801 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4861 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4921 tcccttaacg tgagttttcg ttccactgag tgtcagaccc cgtagaaaag atcaaaggat
     4981 cttc
//
