LOCUS       sequence-328786-        8991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cargo vector for the phiC31 system with Psur5:GFP, cb_unc-119, and 
            Gateway R1R2 site (for gene of interest insertion).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8991)
  AUTHORS   Yang FJ, Chen CN, Chang T, Cheng TW, Chang NC, Kao CY, Lee CC, Huang
            YC, Hsu JC, Li J, Lu MJ, Chan SP, Wang J
  TITLE     phiC31 integrase for recombination-mediated single-copy insertion 
            and genome manipulation in Caenorhabditis elegans.
  JOURNAL   Genetics. 2022 Feb 4;220(2):iyab206. doi: 10.1093/genetics/iyab206.
  PUBMED    34791215
REFERENCE   2  (bases 1 to 8991)
  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: "Genetics.";
            date: "2022-02-4"; volume: "220(2):iyab206. doi"; pages: " 
            10.1093/genetics/iyab206"
COMMENT     Sequence Label: pBRC_double_attB_GFP_donor
FEATURES             Location/Qualifiers
     source          1..8991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    21..54
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     primer_bind     complement(1218..1246)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1994..2014
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             complement(2225..2236)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(4568..4873)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(4670..4689)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(5215..5874)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5789..5808
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(5928..5934,5935..5952,5953..5958))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(5983..6107)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    6359..6392
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     primer_bind     complement(6431..6453)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6553..6572
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6650..6668)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6736..6840
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6841..6909,6910..7701)
                     /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(7059..7078)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7872..8460
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8361..8380
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8614..8631
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8748..8769
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8784..8789,8790..8807,8808..8814)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8822..8838
                     /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     8827..8849
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8846..8862
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8846..8862
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atncaagctt gggctgcagg gtgccagggc gtgcccttgg gctccccggg cgcgtagagt
       61 ggagagtgta gtccctcgtc ggctctctgt gtatttcttt caatgcctgt gtctcggtct
      121 atctttgtct ctatctgttt taaaagtttt ttgcaaagtt tcaatttctt ttttattttc
      181 tcttcttttt ttgcgcattt atttttttta aagagcgctg ttcttcaatt ggaaaacagt
      241 ataacgatcg aaataaaaaa aaaacttgaa atagtatcgg aattgagatg ggggcaattg
      301 aaattgaaga cgcaacaaaa acatgttttt atcgtctgaa attatctaaa ctttgaatct
      361 aacgtgtcaa tggctacttt tacagtagac tttttagagt aatgttaaaa aggataatat
      421 aggtttttct tcaaactatt ttgtcacgcc gcatatattc ttttcatatt ccattcgcaa
      481 agtgcatgat gtaacaaaaa tgaaacgtca taatttttta atctttcaca ctttttttgc
      541 cggccgttaa atcaaaatca aatcaactta atccaacacg tgcgggcttg caattttatt
      601 acaccggtgt cctacagcgc catgttatct tctttgatta gagatctctc acgcaacatt
      661 tgacattgaa aattttgtct ccgaattgtt gtgtacctac tttgtcccga tgtcccaaat
      721 tttcactgat aacaagagat atctcaactg ttcttatcac gttgatacgt agaacgtttt
      781 acactaaacc aaattgcagt ccccttctac gaaccgtttg ctaattgttt tgcatagaac
      841 agtgatcaat gtgttgtgct cactagtcaa tgatgttcgt ctctcatttt tcctacaact
      901 tcactgaaaa ggttttgaat atctggtgat tgtaggcaat cacaccacag ttaatggcct
      961 ggcaaataat gataagcgtg tgataaacct cacaaaaata cgcctagaag tggtgatatc
     1021 gctagttttg tgtcctttcg atttttttcg tgttgctttt ttctgtggat aaacctttga
     1081 actttacatt ttgttttcag aataaccgct ctagaggatc ctatcgattc gcggccgctg
     1141 tacacccggg tgccctatag tgagtcgtat tggtaccggt agaaaaaatg agtaaaggag
     1201 aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaatgggc
     1261 acaaattttc tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa cttaccctta
     1321 aatttatttg cactactgga aaactacctg ttccatgggt aagtttaaac atatatatac
     1381 taactaaccc tgattattta aattttcagc caacacttgt cactactttc tgttatggtg
     1441 ttcaatgctt ctcgagatac ccagatcata tgaaacagca tgactttttc aagagtgcca
     1501 tgcccgaagg ttatgtacag gaaagaacta tatttttcaa agatgacggg aactacaaga
     1561 cacgtaagtt taaacagttc ggtactaact aaccatacat atttaaattt tcaggtgctg
     1621 aagtcaagtt tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta
     1681 aagaagatgg aaacattctt ggacacaaat tggaatacaa ctataactca cacaatgtat
     1741 acatcatggc agacaaacaa aagaatggaa tcaaagttgt aagtttaaac atgattttac
     1801 taactaacta atctgattta aattttcaga acttcaaaat tagacacaac attgaagatg
     1861 gaagcgttca actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc
     1921 ttttaccaga caaccattac ctgtccacac aatctgccct ttcgaaagat cccaacgaaa
     1981 agagagacca catggtcctt cttgagtttg taacagctgc tgggattaca catggcatgg
     2041 acgaactata caaaagccca caagctttgg aattcgcttt gagccaattt atccaagtcc
     2101 ttgtaaaaag tattcgaaaa ttgttaacgg ataaaatgtt tattataata tcaaaaacaa
     2161 tttgtcagtt gaccactttt tgatatattt gacagaaacc gggatgaatt ggctcaaagt
     2221 tagggcgccc ttctattaca gggtttctga taacaaaccg gttattaact cccaacaagg
     2281 gatgatttca attcatcatg ctcaaattga cccaaattaa gttacatgac aaattcatcg
     2341 cccttttcaa ctcttcttgg ctcatcatct gttattctgt tcctattctg tctgcaccct
     2401 ataccccttt gcatactctc tcgtcattcc tttcttggat agtgtcttct tctccccagc
     2461 tctgctactt ctatgacttg cgcccgctgc ttttccgctc gttcctctcc tctctgacgt
     2521 cttcttctgc ttcttttctc cttccttttt cccttctcaa tcttcctctt tttcccattt
     2581 ctgtcaatca ttcgaagaag aagaagaaga agaccctcat tcatttcatt tttttttctg
     2641 tcggtgtgtg ctgctcggtt aagagtgagc tctctattcc acgtcttctt ctttttcttc
     2701 ttcgattcga atcaatcact ccaccaaaac gcattcgttt ttgggattca ccccgcggtt
     2761 cgcaacaggt ttctttttca aattattagc gttataaata gaaaaatggt ggagtttcaa
     2821 taaaaatgat aatttacaaa gtgtattttg attacatgta ctcaaaaagg ttgaaatttt
     2881 tcataccagt tttccggaaa tccatctgat atcattatcg tatttttctt tttaaaaatg
     2941 ttttcaaaaa aaaacaaaat atagctggtt atttggcacc ctctaattac cattttcctg
     3001 tcacaccaca ctcttttcct tcttccactt ctttccgttt tcagccgctt ccaaccaaac
     3061 cgatatgaaa gccgagcaac aacgatcgat tccacccggc tcggcgacct tcccgtcgca
     3121 ggtgagactc agaaaactag agaaaccgct caactaactc ttgatatccg atttcattct
     3181 tttctctttt ctttttgttg aacttttcca tatttccaga tgccacggcc accaccaagc
     3241 accgaacaag gaatcacaac ggaatcggag cttgcgaaga aagctcaaat cactccgaac
     3301 gacgttttag cacttccggg aatcactcaa ggtatcttgc tccctttgga tttcagttta
     3361 aaacataaat ttaggattct tatgctcccc atctgcgaac atctataaca tcgagttcac
     3421 caagttccaa atccgtgatc tggacactga gcaagtgctt ttcgagatcg ccaaaccgga
     3481 gaacgatcag gagaatgatg agtcgccaca ggagtcggca agatacgtgc gttatagatt
     3541 tgctccaaac tttttgaaac tcaaaacggt cggagcaact gtggaattca aagtaggaga
     3601 catcccaatc catcatttcc gaatgatcga acgtcacttc ttcaaagatc gccttctgaa
     3661 gtgttttgac tttgaattcg gattctgtat tccgaattca cgaaacaact gtgaacatat
     3721 ctatgagttc cctcaactct ctcaacaact cagtgagtca ttattctaaa aagtacaatt
     3781 caaaagacta atctcttttt cagtggacga catgatcaac aatccaaacg agactcgttc
     3841 tgacagcttc tatttcgtcg ataacaaact cgtcatgcac aacaaagccg actactcata
     3901 tgatgcataa atatttaata caaaaatgtt ctggataatt attctgtcga atagaaaaaa
     3961 aactccaaat gtgattaaat tccaataatt cctgtctagt ttgttccttc cccttcccct
     4021 tctcatgttc aatgcattcc taagcttttc agttcccccc ttgtttctat attttttcgc
     4081 tgtcctgtca cactcgctaa aaacactaat cacacggaaa tctgttttca ataaaaactc
     4141 caactttaac tcattttcaa tttcaactga aagatttttt cattagagaa tgtcctagcc
     4201 ggccatacaa gtaatccgga tgatcgacgc caacgtcgtt gaattttcaa attttaaata
     4261 ctgaatattt gttttttttc ctattattta tttattctct ttgtgttttt tttcttgctt
     4321 tctaaaaaat taattcaatc caaatctaaa catttttttt tctctttccg tctcccaatt
     4381 cgtattccgc tcctctcatc tgaacacttt gtacaagaaa gctgaacgag aaacgtaaaa
     4441 tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta cataatactg
     4501 taaaacacaa catatccagt cactatggtc gacctgcaga ctggctgtgt ataacggagc
     4561 ctgacattta tattccccag aacatcaggt taatggcgtt tttgatgtca ttttcgcggt
     4621 ggctgagatc agccacttct tccccgataa cggagaccgg cacactggcc atatcggtgg
     4681 tcatcatgcg ccagctttca tccccgatat gcaccaccgg gtaaagttca cgggagactt
     4741 tatctgacag cagacgtgca ctggccaggg ggatcaccat ccgtcgcccg ggcgtgtcaa
     4801 taatatcact ctgtacatcc acaaacagac gataacggct ctctctttta taggtgtaaa
     4861 ccttaaactg catttcacca gtccctgttc tcgtcagcaa aagagccgtt catttcaata
     4921 aaccgggcga cctcagccat cccttcctga ttttccgctt tccagcgttc ggcacgcaga
     4981 cgacgggctt cattctgcat ggttgtgctt accagaccgg agatattgac atcatatatg
     5041 ccttgagcaa ctgatagctg tcgctgtcaa ctgtcactgt aatacgctgc ttcatagcac
     5101 acctcttttt gacatacttc gggtatacat atcagtatat attcttatac cgcaaaaatc
     5161 agcgcgcaaa tacgcatact gttatctggc ttttagtaag ccggatccac gcgtttacgc
     5221 cccgccctgc cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa
     5281 gccatcacag acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg
     5341 cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt
     5401 taaatcaaaa ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat
     5461 aaacccttta gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat
     5521 gtgtagaaac tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt
     5581 ttgctcatgg aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc
     5641 tttcattgcc atacggaatt ccggatgagc attcatcagg cgggcaagaa tgtgaataaa
     5701 ggccggataa aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag
     5761 ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt
     5821 acgatgccat tgggatatat caacggtggt atatccagtg atttttttct ccattttagc
     5881 ttccttagct cctgaaaatc tcgccggatc ctaactcaaa atccacacat tatacgagcc
     5941 ggaagcataa agtgtaaagc ctggggtgcc taatgcggcc gccatagtga ctggatatgt
     6001 tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata
     6061 tttatatcat tttacgtttc tcgttcagct tttttgtaca aacttgtcac aatgtgcaag
     6121 tttatttatc ttctcgcttt catttcatta ggacgtgggg ggaattggtg gaagggggaa
     6181 acacacaaaa ggatgatgga aatgaaataa ggacacacaa tatgcaacaa cattcaattc
     6241 agaaatatgg aggaaggttt aaaagaaaac ataaaaatat atagaggagg aaggaaaact
     6301 agtaaaaaat aagcaaagaa attaggcgaa cgatgagaat tgtcctcgct tgggcctacg
     6361 cgcccgggga gcccaagggc acgccctggc acccctttcg tctcgcgcgt ttcggtgatg
     6421 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     6481 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     6541 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
     6601 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg gccttaaggg cctcgtgata
     6661 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     6721 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     6781 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     6841 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     6901 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     6961 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     7021 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     7081 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     7141 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     7201 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     7261 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     7321 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     7381 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     7441 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     7501 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     7561 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     7621 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     7681 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     7741 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     7801 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     7861 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     7921 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     7981 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     8041 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     8101 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     8161 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     8221 gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg
     8281 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     8341 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     8401 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     8461 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     8521 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     8581 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     8641 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     8701 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     8761 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     8821 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctg
     8881 taagtttaaa catgatctta ctaactaact attctcattt aaattttcag agcttaaaaa
     8941 tggctgaaat cactcacaac gatggatacg ctaacaactt ggaaatgaaa t
//
