LOCUS       sequence-307910-        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      348..936
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     837..856
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1090..1107
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1224..1245
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1260..1265,1266..1283,1284..1290)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1298..1314
                     /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     1303..1325
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1322..1338
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1322..1338
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(3967..4000)
                     /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(5123..5142)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5242..5264
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5302..5320)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5388..5492
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5493..5561,5562..6176,1..177)
                     /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(5711..5730)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
       61 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
      121 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
      181 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
      241 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
      301 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
      361 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
      421 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
      481 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
      541 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
      601 aagtagtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
      661 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
      721 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
      781 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
      841 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
      901 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
      961 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     1021 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     1081 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     1141 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     1201 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     1261 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     1321 acaggaaaca gctatgacca tgattacgcc aagctcactc gtttaggcta ttcccctatt
     1381 tttgatattc cttcgcacat atgaaaacta ctttttttcg aaactgttaa ctccagaatt
     1441 tataaatcta tagcccttac ttgattattt attatcatgg ttactcacct catgtctctt
     1501 tctcttttca agaagtcttc gttcatatct gttgtctctt tcctcctttt gaatctctgc
     1561 tttgctcttc gccattgttt cctgaaaata atgtaacttg aattgtgtaa tatacttttt
     1621 taatttgaat ttggcttgta acgcggaatc actacgtgcg ggatcatttc ttactagaaa
     1681 acccagaaaa tgccatattt cacttatctc ggggtcattt ctaattagaa aagctacaaa
     1741 aaccatttct aattacggtg actttagaac tggtcgaaat agctaattag aaatgggttt
     1801 ttgctaatta gaggtgactc tatgttatct gagtaagcct cgtttgtatg taaactcaca
     1861 gtactaataa atgcaagaca cccgggtttg tctagatatg aaataattga aatatcaatt
     1921 ctgacagaca ataatggtaa tcttgataag gagttccacg cccaggagaa cacgttagtt
     1981 ttcttgtttt tgattgcgtg cgttattttg gagaaaaact cgatttttta caaaataatt
     2041 ttttgaaagg aacactgttc aataagtttt gtcttttttc tcagttgtga tacggttttt
     2101 tattcttttt tgtagttata cagaagaccg cctgcaggat ttgagtagag cactcgaggg
     2161 gagaaaatgc ggagagaaga acccagcatc ttaataaaat tatataattt tgaataaaat
     2221 caaattaaaa ataagattaa ttgaaacaca acaatagaat gcagaaaatt actcataaga
     2281 aataatttat tttgaaagaa gttgtgtcga tttacgataa aaactacacg caaacgaggc
     2341 tccgcgttta caccgtttgg caatggagcg cgcttgctat tagattttag gccagctcaa
     2401 cgaaagttta atttattttt cttctttttt tccatttttt tgagtaaaat atcgaatttt
     2461 ttgtattaat tctgtaaatt ttataatttg caactatttt ggcgtattct gcttgttttt
     2521 atcagcgaat ttttatgcat ttcagtcaaa agtttgagca ggaaaaatga catttaggac
     2581 tttgtaaaat tgtattctta aatttgcttg aaaacctacg tgtacaattg attttattga
     2641 atttttctta cgtttcccgt tgtatcttat tgtatttaac tttcatcata aaataattta
     2701 tttccaggta aaatatccga cgctctcgtg atggtctccg agctcattaa agaaaacatg
     2761 catatgaagc tctacatgga gggaaccgtc aacaaccacc acttcaagtg cacctccgag
     2821 ggagagggaa agccatacga gggaacccaa accatgcgta tcaaggccgt cgagggagga
     2881 ccactcccat tcgccttcga catcctcgcc acctccttca tgtacggatc caaggtaagt
     2941 ttaaacatat atatactaac taaccctgat tatttaaatt ttcagacctt catcaaccac
     3001 acccaaggaa tcccagactt cttcaagcaa tccttcccag agggattcac ctgggagcgt
     3061 gtcaccacct acgaggacgg aggagtcctc accgccaccc aagacacctc cctccaagac
     3121 ggatgcctca tctacaacgt caaggtaagt ttaaacagtt cggtactaac taaccataca
     3181 tatttaaatt ttcagatccg tggagtcaac ttcccatcca acggaccagt catgcaaaag
     3241 aagaccctcg gatgggaggc ctccaccgag accctctacc cagccgacgg aggacttgag
     3301 ggacgtgccg acatggccct caagctcgtc ggaggaggac acctcatctg caacctcaag
     3361 gtaagtttaa acatgatttt actaactaac taatctgatt taaattttca gaccacctac
     3421 cgttccaaga agccagccaa gaacctcaag atgccaggag tctactacgt cgaccgtcgt
     3481 cttgagcgta tcaaggaggc cgacaaggag acctacgtcg agcaacacga ggtcgccgtc
     3541 gcccgttact gcgacctccc atccaagctc ggacaccgtt aagtccaatt actcttcaac
     3601 atccctacat gctctttctc cctgtgctcc caccccctat ttttgttatt atcaaaaaac
     3661 ttctcttaat ttctttgttt tttagcttct tttaagtcac ctctaacaat gaaattgtgt
     3721 agattcaaaa atagaattaa ttcgtaataa aaagtcgaaa aaaattgtgc tccctccccc
     3781 cattaataat aattctatcc caaaatctac acaatgttct gtgtacactt cttatgtttt
     3841 ttacttctga taaatttttt tgaaacatca tagaaaaaac cgcacacaaa ataccttatc
     3901 atatgttacg tttcagttta tgaccgcaat ttttacttaa gttcaatccg gaaaggtaag
     3961 tttaaaatga cttcgtatag catacattat acgaagttat tttcagggag agcaaaagat
     4021 ttttgctttc gtcgtaaatc tacacacgcg tctcttccgt gcgagagtcc aagccagcag
     4081 ccaaattcgt tgactgagta ttcaacgttt atacgttgtc ggcaacgaga aataggaaaa
     4141 tgcatcggga aatgttcttt tttcgatttt ttccaaggtt ttgacaaatt ttaccacgaa
     4201 ttttgctatg ttttcaatta aaaaatatgt tattcaactg tttctatgag gaaaataagg
     4261 ctttgcatgt aattttctta ttcagcataa tttttaatta atttgaattt tctgtcctaa
     4321 cgtttatttt gttttcttgg ttatgactga tctgaaatta atttttgaat tttaaggtaa
     4381 tatgaaaaaa cccgagttga ccgccacatc cgtagagaag ttcctcatcg agaagttcga
     4441 ctccgtctcc gacctcatgc aactctccga gggagaggag tcccgtgcct tctccttcga
     4501 cgtcggagga cgtggatacg tcctccgtgt caactcctgc gccgacggat tctacaagga
     4561 ccgttacgtc taccgtcact tcgcctccgc cgccctccca atcccagagg tcctcgacat
     4621 cggagagttc tccgagtccc tcacctactg catctcccgt cgtgcccaag gagtcaccct
     4681 ccaagacctc ccagagaccg agctcccagc cgtcctccaa ccagtcgccg aggccatgga
     4741 cgccatcgcc gccgccgacc tctcccaaac ctccggattc ggaccattcg gaccacaagg
     4801 aatcggacaa tacaccacct ggcgtgactt catctgcgcc atcgccgacc cacacgtcta
     4861 ccactggcaa accgtcatgg acgacaccgt ctccgcctcc gtcgcccaag ccctcgacga
     4921 gctcatgctc tgggccgagg actgcccaga ggtccgtcac ctcgtccacg ccgacttcgg
     4981 atccaacaac gtcctcaccg acaacggacg tatcaccgcc gtcatcgact ggtccgaggc
     5041 catgttcgga gactcccaat atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     5101 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     5161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     5221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     5281 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     5341 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5401 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5461 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5521 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5581 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5641 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5701 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5761 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5821 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5881 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5941 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6001 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6061 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6121 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggttt
//
