LOCUS       sequence-486674-        6648 bp    DNA     circular SYN 18-SEP-2025
DEFINITION  Selectable donor plasmid for insertion by Bxb1 integrase into 
            pre-inserted attB landing pad site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6648)
  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 6648)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 18, 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: pCMV Bxb1 attP dsRed Hygro
FEATURES             Location/Qualifiers
     source          1..6648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     611..630
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     689..708
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     810..826
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     810..826
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     831..850
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        831..849
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(905..1028)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     enhancer        1161..1464
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1465..1668
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             1797..2834
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
                     MGEAN"
     misc_feature    join(2847..3421,3422..3424,3425..3433)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3014..3031)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3241..3260
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3434..4111
                     /codon_start=1
                     /product="noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2008)"
                     /label=DsRed-Express2
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
                     ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(3614..3634)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3832..3851
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4026..4049
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4233..4314
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4270..4289)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    4350..4407
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attP site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     promoter        complement(4439..4457)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4440..4457)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(4573..4776)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        4777..5080
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(5099..5121)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5110..5126
                     /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(5134..5140,5141..5158,5159..5164))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(5191..5209)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5277..5381
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    5482..5576
                     /label=5X UAS
                     /bound_moiety="GAL4"
                     /note="five tandem copies of the ""ScaI site"" 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     primer_bind     complement(5630..5647)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5801..6389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5881..5900)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6536..6648,1..697))
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
       61 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
      121 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
      181 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
      241 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
      301 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
      361 caggatattc ttctaatacc tggaatgctg tttttccggg gatcgcagtg gtgagtaacc
      421 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
      481 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
      541 tcagaaacaa ctctggcgca tcgggcttcc catacaagcg atagattgtc gcacctgatt
      601 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
      661 atcgcggcct cgacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
      721 tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc
      781 agagattttg agacacgggc cagagctgcc aggaaacagc tatgaccatg taatacgact
      841 cactataggg gatatcgcgg ccgccctgca gtacaggtca ctaataccat ctaagtagtt
      901 gattcatagt gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa
      961 aatctaattt aatatattga tatttatatc attttacgtt tctcgttcaa ctttattata
     1021 caaagttgtg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     1081 accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc attagttcat
     1141 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     1201 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     1261 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     1321 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     1381 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     1441 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     1501 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     1561 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     1621 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac
     1681 cgtcagatcc gctagcgcta ccggactcag atctcgagct caagcttcga attctgcagt
     1741 cgacggtacc gcgggcccgg gaacagcgtg ccgcagatcc cggggggcaa tgagatatga
     1801 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     1861 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     1921 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
     1981 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
     2041 aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
     2101 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
     2161 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
     2221 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
     2281 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
     2341 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
     2401 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     2461 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     2521 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     2581 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     2641 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     2701 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     2761 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccggat cgggagatgg
     2821 gggaggctaa ctgaacccgg gatccgcccc tctccctccc ccccccctaa cgttactggc
     2881 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg
     2941 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
     3001 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
     3061 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
     3121 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
     3181 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
     3241 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
     3301 atgggatctg atctggggcc tcggtacaca tgctttacat gtgtttagtc gaggttaaaa
     3361 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa
     3421 tatggccaca accatggata gcactgagaa cgtcatcaag cccttcatgc gcttcaaggt
     3481 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggcaa
     3541 gccctacgag ggcacccaga ccgccaagct gcaggtgacc aagggcggcc ccctgccctt
     3601 cgcctgggac atcctgtccc cccagttcca gtacggctcc aaggtgtacg tgaagcaccc
     3661 cgccgacatc cccgactaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     3721 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     3781 caccttcatc taccacgtga agttcatcgg cgtgaacttc ccctccgacg gccccgtaat
     3841 gcagaagaag actctgggct gggagccctc caccgagcgc ctgtaccccc gcgacggcgt
     3901 gctgaagggc gagatccaca aggcgctgaa gctgaagggc ggcggccact acctggtgga
     3961 gttcaagtca atctacatgg ccaagaagcc cgtgaagctg cccggctact actacgtgga
     4021 ctccaagctg gacatcacct cccacaacga ggactacacc gtggtggagc agtacgagcg
     4081 cgccgaggcc cgccaccacc tgttccagta ggcggccgcg actctagatc ataatcagcc
     4141 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     4201 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     4261 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4321 gttgtggttt gtccaaactc agcggccgcg tcggggtttg taccgtacac cactgagacc
     4381 gcggtggttg accagacaaa ccacgacgaa ttccggaccg gtacctgcag gcgtaccttc
     4441 tatagtgtca cctaaatagc tttttgcaaa agcctaggct agagtccgga ggctggatcg
     4501 gtcccggtgt cttctatgga ggtcaaaaca gcgtggatgg cgtctccagg cgatctgacg
     4561 gttcactaaa cgagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg
     4621 cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa
     4681 caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta
     4741 tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac
     4801 gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc
     4861 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     4921 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc
     4981 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     5041 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg acctgcacga tgctgtttcc
     5101 tgtgtgaaat tgttatccgc tcacaattcc acacattata cgagccggaa gcataaagtg
     5161 taaagcctgg ggtgcctaat gagtgaaagg gcctcgtgat acgcctattt ttataggtta
     5221 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     5281 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5341 aaccctgata aatgcttcaa taatattgaa aaagcgcgaa ttgcaagctc tgcattaatg
     5401 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg ctgagctctc
     5461 cggcgctcgc tagagtctcc gctcggagga cagtactccg ctcggaggac agtactccgc
     5521 tcggaggaca gtactccgct cggaggacag tactccgctc ggaggacagt actccgacct
     5581 gcaggcatgg aagcttggat cgagctctca atacttacaa actcctttcc tcgctcactg
     5641 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5701 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5761 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5821 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5881 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5941 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6001 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6061 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6121 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6181 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6241 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6301 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6361 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6421 acgctcagtg gaacgacgcg taactcacgt taagggattt tggtcatgag cttgcgccgt
     6481 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattaga
     6541 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     6601 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggc
//
