LOCUS       sequence-486641-        5883 bp    DNA     circular SYN 17-SEP-2025
DEFINITION  Mammalian expression of hyperactive Bxb1 Integrase mutant (I87L, 
            A369P, 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-c22
FEATURES             Location/Qualifiers
     source          1..5883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      3..457
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(90..109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     299..320
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             join(744..812,813..1604)
                     /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(962..981)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     protein_bind    1706..1739
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      1817..2405
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2306..2325
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2559..2576
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        complement(2650..2754)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2822..2840
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2867..2872,2873..2890,2891..2897)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    2905..2921
                     /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     2910..2932
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     enhancer        2951..3254
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3255..3458
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3409..3429
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3455..3479
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        3574..3592
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3574..3591
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5220..5239)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5221..5239)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    5626..5760
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5663..5682)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5717..5736
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
       61 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
      121 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
      181 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
      241 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tcttaatagt
      301 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
      361 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
      421 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
      481 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatgc
      541 caggtcttgg actggtgaga acggcttgct cggcagcttc gatgtgtgct ggagggagaa
      601 taaaggtcta agatgtgcga tagagggaag tcgcattgaa ttatgtgctg tgtagggatc
      661 gctggtatca aatatgtgtg cccacccctg gcatgagaca ataaccctga taaatgcttc
      721 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
      781 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
      841 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
      901 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
      961 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     1021 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     1081 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     1141 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     1201 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     1261 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     1321 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     1381 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     1441 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     1501 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     1561 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     1621 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     1681 agatcctttt tgataatctc atgccataac ttcgtataat gtatgctata cgaagttatg
     1741 gcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1801 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1861 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1921 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     1981 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2041 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2101 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2161 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2221 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2281 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2341 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2401 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2461 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2521 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2581 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     2641 gagcttgcaa ttcgcgcttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     2701 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     2761 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     2821 aaataggcgt atcacgaggc cctttcactc attaggcacc ccaggcttta cactttatgc
     2881 ttccggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca ggaaacagca
     2941 tcgtgcaggt cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     3001 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     3061 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     3121 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     3181 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     3241 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     3301 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     3361 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     3421 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg
     3481 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
     3541 tccggactct agcctaggct tttgcaaaaa gctatttagg tgacactata gaaggtacgc
     3601 ctgcaggtac cggtccggaa ttcccgggtc gacgagctca ctagtgccgc caccatgcga
     3661 gccctcgtag tcatacgcct ttctcgcgta accgatgcga cgactagtcc cgagagacaa
     3721 ttggagtctt gtcaacaatt gtgtgcgcag cgaggctggg acgtcgtagg agtcgcagag
     3781 gaccttgatg tctcaggagc cgttgatcct tttgacagga aaaggcgacc aaatcttgcg
     3841 agatggcttg cttttgagga gcagccattt gatgttatag tagcatatag ggtcgacaga
     3901 ttgacgcggt ctctacgaca tctccagcag ctggtccatt gggccgaaga ccacaagaaa
     3961 ctggttgtga gcgcaacgga ggcacacttt gatacaacta ctcctttcgc ggctgttgtg
     4021 atcgcgctca tgggtaccgt ggctcaaatg gagcttgagg caatcaaaga acggaatcga
     4081 tcagctgccc attttaatat aagagctggt aaatatcgcg gctctctgcc tccctggggg
     4141 tatcttccga caagagtaga cggcgaatgg cgcctcgttc cggacccggt acaacgggag
     4201 aggatactgg aagtatatca ccgagtggta gacaatcacg agccgcttca cttggtcgcg
     4261 cacgatttga atagacgggg cgtactttct cctaaagatt atttcgcgca actgcaaggg
     4321 cgcgaaccgc aaggtagaga gtggagtgca acagccctca agagatcaat gatctcagag
     4381 gctatgctgg ggtacgcgac gctcaatggc aaaacagtgc gagacgatga tggtgccccc
     4441 ctcgttagag ccgaacctat cctgactcgg gaacaactcg aggccttgcg ggccgagctg
     4501 gtgaaaactt cacgcgccaa accagctgtt tcaacaccct cacttcttct gcgagttctg
     4561 ttctgtgcag tctgcggaga gccggcgtat aaatttgcag gtggtgggag aaagcatccg
     4621 aggtatcgat gccgaagcat ggggtttccc aaacattgcg gcaatggcac cgtcgctatg
     4681 gccgagtggg atgcattttg tgaggaacag gtattggacc ttttggggga cgcagagagg
     4741 ttggagaagg tttgggtccc aggatccgat tctgcggtgg agcttgccga agtcaacgct
     4801 gagctcgtgg acctcacgtc cctcattggt agcccagcgt accgcgcggg ttcccctcag
     4861 agagaggcgc ttgatgcccg catcgccgct cttgcggcca gacaagagga gcttgaggga
     4921 ctggaggcga ggccaagcgg ttgggagtgg aggggaacgg gccagcgatt cggcgattgg
     4981 tggcgagagc aggacacagc tgcgaagaac acttggctta gatccatgaa tgttcggttg
     5041 acctttgatg tgaggggcgg tctgacgcga acgatcgact tcggcgacct tcaagagtac
     5101 gaacagcatc ttaggctggg gtcagtagtc gagcgcttgc acaccgggat gtcgtagcga
     5161 gacacccgga tccctcgagg ggcccaagct tacgcgtgca tgcgacgtca tagctctctc
     5221 cctatagtga gtcgtattat aagctagctt gggatctttg tgaaggaacc ttacttctgt
     5281 ggtgtgacat aattggacaa actacctaca gagatttaaa gctctaaggt aaatataaaa
     5341 tttttaagtg tataatgtgt taaactagct gcatatgctt gctgcttgag agttttgctt
     5401 actgagtatg atttatgaaa atattataca caggagctag tgattctaat tgtttgtgta
     5461 ttttagattc acagtcccaa ggctcatttc aggcccctca gtcctcacag tctgttcatg
     5521 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     5581 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     5641 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     5701 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     5761 gatcctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttggctggcg
     5821 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     5881 atg
//
