LOCUS       sequence-486636-        5883 bp    DNA     circular SYN 17-SEP-2025
DEFINITION  Mammalian expression of hyperactive Bxb1 Integrase mutant (I87L, 
            E434G).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5883)
  AUTHORS   Hew BE, Gupta S, Sato R, Waller DF, Stoytchev I, Short JE, Sharek L,
            Tran CT, Badran AH, Owens JB
  TITLE     Directed evolution of hyperactive integrases for site specific 
            insertion of transgenes.
  JOURNAL   Nucleic Acids Res. 2024 Jul 2:gkae534. doi: 10.1093/nar/gkae534.
  PUBMED    38953167
REFERENCE   2  (bases 1 to 5883)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2024 Jul 2:gkae534. doi: 10.1093/nar/gkae534."
COMMENT     Sequence Label: pCMV2 Bxb1-c10
FEATURES             Location/Qualifiers
     source          1..5883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             join(743..811,812..1603)
                     /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(961..980)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    1705..1738
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      1816..2404
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2305..2324
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2558..2575
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        complement(2649..2753)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2821..2839
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2866..2871,2872..2889,2890..2896)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    2904..2920
                     /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     2909..2931
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     enhancer        2950..3253
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3254..3457
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3408..3428
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3454..3478
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        3573..3591
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3573..3590
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5219..5238)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5220..5238)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    5625..5759
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5662..5681)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5716..5735
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt cttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgcc
      541 aggtcttgga ctggtgagaa cggcttgctc ggcagcttcg atgtgtgctg gagggagaat
      601 aaaggtctaa gatgtgcgat agagggaagt cgcattgaat tatgtgctgt gtagggatcg
      661 ctggtatcaa atatgtgtgc ccacccctgg catgagacaa taaccctgat aaatgcttca
      721 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
      781 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
      841 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
      901 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
      961 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     1021 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     1081 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     1141 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     1201 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     1261 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     1321 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     1381 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     1441 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     1501 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     1561 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     1621 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     1681 gatccttttt gataatctca tgccataact tcgtataatg tatgctatac gaagttatgg
     1741 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1801 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1861 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1921 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     1981 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2041 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2101 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2161 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2221 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2281 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2341 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2401 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2461 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2521 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2581 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     2641 agcttgcaat tcgcgctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     2701 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     2761 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     2821 aataggcgta tcacgaggcc ctttcactca ttaggcaccc caggctttac actttatgct
     2881 tccggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcat
     2941 cgtgcaggtc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     3001 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     3061 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     3121 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     3181 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     3241 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     3301 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     3361 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     3421 taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc
     3481 ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct
     3541 ccggactcta gcctaggctt ttgcaaaaag ctatttaggt gacactatag aaggtacgcc
     3601 tgcaggtacc ggtccggaat tcccgggtcg acgagctcac tagtgccgcc accatgcgag
     3661 ccctcgtagt catacgcctt tctcgcgtaa ccgatgcgac gactagtccc gagagacaat
     3721 tggagtcttg tcaacaattg tgtgcgcagc gaggctggga cgtcgtagga gtcgcagagg
     3781 accttgatgt ctcaggagcc gttgatcctt ttgacaggaa aaggcgacca aatcttgcga
     3841 gatggcttgc ttttgaggag cagccatttg atgttatagt agcatatagg gtcgacagat
     3901 tgacgcggtc tctacgacat ctccagcagc tggtccattg ggccgaagac cacaagaaac
     3961 tggttgtgag cgcaacggag gcacactttg atacaactac tcctttcgcg gctgttgtga
     4021 tcgcgctcat gggtaccgtg gctcaaatgg agcttgaggc aatcaaagaa cggaatcgat
     4081 cagctgccca ttttaatata agagctggta aatatcgcgg ctctctgcct ccctgggggt
     4141 atcttccgac aagagtagac ggcgaatggc gcctcgttcc ggacccggta caacgggaga
     4201 ggatactgga agtatatcac cgagtggtag acaatcacga gccgcttcac ttggtcgcgc
     4261 acgatttgaa tagacggggc gtactttctc ctaaagatta tttcgcgcaa ctgcaagggc
     4321 gcgaaccgca aggtagagag tggagtgcaa cagccctcaa gagatcaatg atctcagagg
     4381 ctatgctggg gtacgcgacg ctcaatggca aaacagtgcg agacgatgat ggtgcccccc
     4441 tcgttagagc cgaacctatc ctgactcggg aacaactcga ggccttgcgg gccgagctgg
     4501 tgaaaacttc acgcgccaaa ccagctgttt caacaccctc acttcttctg cgagttctgt
     4561 tctgtgcagt ctgcggagag ccggcgtata aatttgcagg tggtgggaga aagcatccga
     4621 ggtatcgatg ccgaagcatg gggtttccca aacattgcgg caatggcacc gtcgctatgg
     4681 ccgagtggga tgcattttgt gaggaacagg tattggacct tttgggggac gcagagaggt
     4741 tggagaaggt ttgggtcgca ggatccgatt ctgcggtgga gcttgccgaa gtcaacgctg
     4801 agctcgtgga cctcacgtcc ctcattggta gcccagcgta ccgcgcgggt tcccctcaga
     4861 gagaggcgct tgatgcccgc atcgccgctc ttgcggccag acaagaggag cttgagggac
     4921 tggaggcgag gccaagcggt tgggagtgga ggggaacggg ccagcgattc ggcgattggt
     4981 ggcgagagca ggacacagct gcgaagaaca cttggcttag atccatgaat gttcggttga
     5041 cctttgatgt gaggggcggt ctgacgcgaa cgatcgactt cggcgacctt caagagtacg
     5101 aacagcatct taggctgggg tcagtagtcg agcgcttgca caccgggatg tcgtagcgag
     5161 acacccggat ccctcgaggg gcccaagctt acgcgtgcat gcgacgtcat agctctctcc
     5221 ctatagtgag tcgtattata agctagcttg ggatctttgt gaaggaacct tacttctgtg
     5281 gtgtgacata attggacaaa ctacctacag agatttaaag ctctaaggta aatataaaat
     5341 ttttaagtgt ataatgtgtt aaactagctg catatgcttg ctgcttgaga gttttgctta
     5401 ctgagtatga tttatgaaaa tattatacac aggagctagt gattctaatt gtttgtgtat
     5461 tttagattca cagtcccaag gctcatttca ggcccctcag tcctcacagt ctgttcatga
     5521 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     5581 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     5641 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5701 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg
     5761 atcctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tggctggcgt
     5821 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     5881 tgg
//
