LOCUS       sequence-299735-        6176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insertion of rpl-28p::mKate2::unc-54 3'UTR into a split hygromycin 
            landing pad. Can be modified to insert a different gene(s) of 
            interest..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6176)
  AUTHORS   Stevenson ZC, Moerdyk-Schauwecker MJ, Jamison B, Phillips PC
  TITLE     Rapid Self-Selecting and Clone-Free Integration of Transgenes into 
            Engineered CRISPR Safe Harbor Locations in Caenorhabditis elegans.
  JOURNAL   G3 (Bethesda). 2020 Aug 19. pii: g3.120.401400. doi: 
            10.1534/g3.120.401400.
  PUBMED    32816924
REFERENCE   2  (bases 1 to 6176)
  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: "G3 
            (Bethesda). 2020 Aug 19. pii: g3.120.401400. doi: 
            10.1534/g3.120.401400."
COMMENT     Sequence Label: pMS81(rpl-28p::mKate2::unc-54
            3'UTR::LoxP::rps...
FEATURES             Location/Qualifiers
     source          1..6176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      340..928
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     829..848
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1082..1099
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1216..1237
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1252..1257,1258..1275,1276..1282)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1290..1306
                     /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     1295..1317
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1314..1330
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1314..1330
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(3959..3992)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5115..5134)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5234..5256
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5294..5312)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5380..5484
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5485..5553,5554..6176,1..169)
                     /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(5703..5722)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
       61 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
      121 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
      181 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
      241 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
      301 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
      361 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
      421 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
      481 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
      541 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
      601 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
      661 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
      721 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
      781 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
      841 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
      901 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
      961 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1021 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1081 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     1141 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     1201 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     1261 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     1321 cagctatgac catgattacg ccaagctcac tcgtttaggc tattccccta tttttgatat
     1381 tccttcgcac atatgaaaac tacttttttt cgaaactgtt aactccagaa tttataaatc
     1441 tatagccctt acttgattat ttattatcat ggttactcac ctcatgtctc tttctctttt
     1501 caagaagtct tcgttcatat ctgttgtctc tttcctcctt ttgaatctct gctttgctct
     1561 tcgccattgt ttcctgaaaa taatgtaact tgaattgtgt aatatacttt tttaatttga
     1621 atttggcttg taacgcggaa tcactacgtg cgggatcatt tcttactaga aaacccagaa
     1681 aatgccatat ttcacttatc tcggggtcat ttctaattag aaaagctaca aaaaccattt
     1741 ctaattacgg tgactttaga actggtcgaa atagctaatt agaaatgggt ttttgctaat
     1801 tagaggtgac tctatgttat ctgagtaagc ctcgtttgta tgtaaactca cagtactaat
     1861 aaatgcaaga cacccgggtt tgtctagata tgaaataatt gaaatatcaa ttctgacaga
     1921 caataatggt aatcttgata aggagttcca cgcccaggag aacacgttag ttttcttgtt
     1981 tttgattgcg tgcgttattt tggagaaaaa ctcgattttt tacaaaataa ttttttgaaa
     2041 ggaacactgt tcaataagtt ttgtcttttt tctcagttgt gatacggttt tttattcttt
     2101 tttgtagtta tacagaagac cgcctgcagg atttgagtag agcactcgag gggagaaaat
     2161 gcggagagaa gaacccagca tcttaataaa attatataat tttgaataaa atcaaattaa
     2221 aaataagatt aattgaaaca caacaataga atgcagaaaa ttactcataa gaaataattt
     2281 attttgaaag aagttgtgtc gatttacgat aaaaactaca cgcaaacgag gctccgcgtt
     2341 tacaccgttt ggcaatggag cgcgcttgct attagatttt aggccagctc aacgaaagtt
     2401 taatttattt ttcttctttt tttccatttt tttgagtaaa atatcgaatt ttttgtatta
     2461 attctgtaaa ttttataatt tgcaactatt ttggcgtatt ctgcttgttt ttatcagcga
     2521 atttttatgc atttcagtca aaagtttgag caggaaaaat gacatttagg actttgtaaa
     2581 attgtattct taaatttgct tgaaaaccta cgtgtacaat tgattttatt gaatttttct
     2641 tacgtttccc gttgtatctt attgtattta actttcatca taaaataatt tatttccagg
     2701 taaaatatcc gacgctctcg tgatggtctc cgagctcatt aaagaaaaca tgcatatgaa
     2761 gctctacatg gagggaaccg tcaacaacca ccacttcaag tgcacctccg agggagaggg
     2821 aaagccatac gagggaaccc aaaccatgcg tatcaaggcc gtcgagggag gaccactccc
     2881 attcgccttc gacatcctcg ccacctcctt catgtacgga tccaaggtaa gtttaaacat
     2941 atatatacta actaaccctg attatttaaa ttttcagacc ttcatcaacc acacccaagg
     3001 aatcccagac ttcttcaagc aatccttccc agagggattc acctgggagc gtgtcaccac
     3061 ctacgaggac ggaggagtcc tcaccgccac ccaagacacc tccctccaag acggatgcct
     3121 catctacaac gtcaaggtaa gtttaaacag ttcggtacta actaaccata catatttaaa
     3181 ttttcagatc cgtggagtca acttcccatc caacggacca gtcatgcaaa agaagaccct
     3241 cggatgggag gcctccaccg agaccctcta cccagccgac ggaggacttg agggacgtgc
     3301 cgacatggcc ctcaagctcg tcggaggagg acacctcatc tgcaacctca aggtaagttt
     3361 aaacatgatt ttactaacta actaatctga tttaaatttt cagaccacct accgttccaa
     3421 gaagccagcc aagaacctca agatgccagg agtctactac gtcgaccgtc gtcttgagcg
     3481 tatcaaggag gccgacaagg agacctacgt cgagcaacac gaggtcgccg tcgcccgtta
     3541 ctgcgacctc ccatccaagc tcggacaccg ttaagtccaa ttactcttca acatccctac
     3601 atgctctttc tccctgtgct cccaccccct atttttgtta ttatcaaaaa acttctctta
     3661 atttctttgt tttttagctt cttttaagtc acctctaaca atgaaattgt gtagattcaa
     3721 aaatagaatt aattcgtaat aaaaagtcga aaaaaattgt gctccctccc cccattaata
     3781 ataattctat cccaaaatct acacaatgtt ctgtgtacac ttcttatgtt ttttacttct
     3841 gataaatttt tttgaaacat catagaaaaa accgcacaca aaatacctta tcatatgtta
     3901 cgtttcagtt tatgaccgca atttttactt aagttcaatc cggaaaggta agtttaaaat
     3961 gacttcgtat agcatacatt atacgaagtt attttcaggg agagcaaaag atttttgctt
     4021 tcgtcgtaaa tctacacacg cgtctcttcc gtgcgagagt ccaagccagc agccaaattc
     4081 gttgactgag tattcaacgt ttatacgttg tcggcaacga gaaataggaa aatgcatcgg
     4141 gaaatgttct tttttcgatt ttttccaagg ttttgacaaa ttttaccacg aattttgcta
     4201 tgttttcaat taaaaaatat gttattcaac tgtttctatg aggaaaataa ggctttgcat
     4261 gtaattttct tattcagcat aatttttaat taatttgaat tttctgtcct aacgtttatt
     4321 ttgttttctt ggttatgact gatctgaaat taatttttga attttaaggt aatatgaaaa
     4381 aacccgagtt gaccgccaca tccgtagaga agttcctcat cgagaagttc gactccgtct
     4441 ccgacctcat gcaactctcc gagggagagg agtcccgtgc cttctccttc gacgtcggag
     4501 gacgtggata cgtcctccgt gtcaactcct gcgccgacgg attctacaag gaccgttacg
     4561 tctaccgtca cttcgcctcc gccgccctcc caatcccaga ggtcctcgac atcggagagt
     4621 tctccgagtc cctcacctac tgcatctccc gtcgtgccca aggagtcacc ctccaagacc
     4681 tcccagagac cgagctccca gccgtcctcc aaccagtcgc cgaggccatg gacgccatcg
     4741 ccgccgccga cctctcccaa acctccggat tcggaccatt cggaccacaa ggaatcggac
     4801 aatacaccac ctggcgtgac ttcatctgcg ccatcgccga cccacacgtc taccactggc
     4861 aaaccgtcat ggacgacacc gtctccgcct ccgtcgccca agccctcgac gagctcatgc
     4921 tctgggccga ggactgccca gaggtccgtc acctcgtcca cgccgacttc ggatccaaca
     4981 acgtcctcac cgacaacgga cgtatcaccg ccgtcatcga ctggtccgag gccatgttcg
     5041 gagactccca atatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5101 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     5161 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5221 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5281 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     5341 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5401 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5461 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5521 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5581 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5641 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5701 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5761 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5821 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5881 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5941 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6001 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6061 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6121 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctga
//
