LOCUS       sequence-370778-        6952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite Gateway [4-3] Destination vector; miniMos transposon with 
            lox2272-flanked Cbr-unc-119 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6952)
  AUTHORS   Schwartz ML, Davis MW, Rich MS, Jorgensen EM
  TITLE     High-efficiency CRISPR gene editing in C. elegans using Cas9 
            integrated into the genome.
  JOURNAL   PLoS Genet. 2021 Nov 8;17(11):e1009755. doi: 
            10.1371/journal.pgen.1009755. eCollection 2021 Nov.
  PUBMED    34748534
REFERENCE   2  (bases 1 to 6952)
  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: "PLoS 
            Genet."; date: "2021-11-8"; volume: "17(11):e1009755. doi"; pages: "
            10.1371/journal.pgen.1009755. eCollection 2021 Nov"
COMMENT     Sequence Label: pMLS713
FEATURES             Location/Qualifiers
     source          1..6952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    346..379
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             2338..2349
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    2498..2531
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     2554..2570
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2554..2570
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    2590..2713
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(2754..3059)
                     /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
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(2856..2875)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3379..4059)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     3974..3993
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(4113..4119,4120..4137,4138..4143))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(4168..4291)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4299..4316)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4299..4315)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(4321..4332)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     5002..5024
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5062..5080)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5148..5252
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5253..5321,5322..6113)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGSDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5471..5490)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6284..6872
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6773..6792
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgcac cgtacgtctc
       61 gaggaattct atcaggtgta caagtatgaa atgtcgtttc gaacatatag atgtctcgca
      121 aacgtaaata tttatcgatt gtcataaaac tttgaccttg tgaagtgtca accttgactg
      181 tcgaaccacc atagtttggc gcgaattgag cgtcataatt gtttactctc agtgcagtca
      241 acatgtcgag tttcgtgccg aataaagagc aaacgcggac agtattaatt ttctgttttc
      301 atttgaagaa aacagctgcg gcagtttaaa catgatttta ctaacataac ttcgtataaa
      361 gtatcctata cgaagttatg acattctcta atgaaaaaat ctttcagttg aaattgaaaa
      421 tgagttaaag ttggagtttt tattgaaaac agatttccgt gtgattagtg tttttagcga
      481 gtgtgacagg acagcgaaaa aatatagaaa caagggggga actgaaaagc ttaggaatgc
      541 attgaacatg agaaggggaa agggaaggaa caaactagac aggaattatt ggaatttaat
      601 cacatttgga gttttttttc tattcgacag aataattatc cagaacattt ttgtattaaa
      661 tatttatgca tcatatgagt agtcggcttt gttgtgcatg acgagtttgt tatcgacgaa
      721 atagaagctg tcagaacgag tctcgtttgg attgttgatc atgtcgtcca ctgaaaaaga
      781 gattagtctt ttgaattgta ctttttagaa taatgactca ctgagttgtt gagagagttg
      841 agggaactca tagatatgtt cacagttgtt tcgtgaattc ggaatacaga atccgaattc
      901 aaagtcaaaa cacttcagaa ggcgatcttt gaagaagtga cgttcgatca ttcggaaatg
      961 atggattggg atgtctccta ctttgaattc cacagttgct ccgaccgttt tgagtttcaa
     1021 aaagtttgga gcaaatctat aacgcacgta tcttgccgac tcctgtggcg actcatcatt
     1081 ctcctgatcg ttctccggtt tggcgatctc gaaaagcact tgctcagtgt ccagatcacg
     1141 gatttggaac ttggtgaact cgatgttata gatgttcgca gatggggagc ataagaatcc
     1201 taaatttatg ttttaaactg aaatccaaag ggagcaagat accttgagtg attcccggaa
     1261 gtgctaaaac gtcgttcgga gtgatttgag ctttcttcgc aagctccgat tccgttgtga
     1321 ttccttgttc ggtgcttggt ggtggccgtg gcatctggaa atatggaaaa gttcaacaaa
     1381 aagaaaagag aaaagaatga aatcggatat caagagttag ttgagcggtt tctctagttt
     1441 tctgagtctc acctgcgacg ggaaggtcgc cgagccgggt ggaatcgatc gttgttgctc
     1501 ggctttcata tcggtttggt tggaagcggc tgaaaacgga aagaagtgga agaaggaaaa
     1561 gagtgtggtg tgacaggaaa atggtaatta gagggtgcca aataaccagc tatattttgt
     1621 ttttttttga aaacattttt aaaaagaaaa atacgataat gatatcagat ggatttccgg
     1681 aaaactggta tgaaaaattt caaccttttt gagtacatgt aatcaaaata cactttgtaa
     1741 attatcattt ttattgaaac tccaccattt ttctatttat aacgctaata atttgaaaaa
     1801 gaaacctatt gcgaaccgcg gggtgaatcc caaaaacgaa tgcgttttgg tggagtgatt
     1861 gattcgaatc gaagaagaaa aagaagaaga cgtggaatag agagctcact cttaaccgag
     1921 cagcacacac cgacagaaaa aaaaatgaaa tgaatgaggg tcttcttctt cttcttcttc
     1981 gaatgattga cagaaatggg aaaaagagga agattgagaa gggaaaaagg aaggagaaaa
     2041 gaagcagaag aagacgtcag agaggagagg aacgagcgga aaagcagcgg gcgcaagtca
     2101 tagaagtagc agagctgggg agaagaagac actatccaag aaaggaatga cgagagagta
     2161 tgcaaagggg tatagggtgc agacagaata ggaacagaat aacagatgat gagccaagaa
     2221 gagttgaaaa gggcgatgaa tttgtcatgt aacttaattt gggtcaattt gagcatgatg
     2281 aattgaaatc atcccttgtt gggagttaat aaccggtttg ttatcagaaa ccctgtaata
     2341 gaagggcgcc ctaactttga gccaattcat cccggtttct gtcaaatata tcaaaaagtg
     2401 gtcaactgac aaattgtttt tgatattata ataaacattt tatccgttaa caattttcga
     2461 atacttttta caaggacttg gataaattgg ctcaaagata acttcgtata aagtatccta
     2521 tacgaagtta ttaactaatc tgatttaaat tttcaggaaa cagctatgac catgattacg
     2581 ccaagctatc aactttgtat agaaaagttg aacgagaaac gtaaaatgat ataaatatca
     2641 atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa acacaacata
     2701 tccagtcact atggtcgacc tgcagactgg ctgtgtataa gggagcctga catttatatt
     2761 ccccagaaca tcaggttaat ggcgtttttg atgtcatttt cgcggtggct gagatcagcc
     2821 acttcttccc cgataacgga gaccggcaca ctggccatat cggtggtcat catgcgccag
     2881 ctttcatccc cgatatgcac caccgggtaa agttcacggg ggactttatc tgacagcaga
     2941 cgtgcactgg ccagggggat caccatccgt cgcccgggcg tgtcaataat atcactctgt
     3001 acatccacaa acagacgata acggctctct cttttatagg tgtaaacctt aaactgcatt
     3061 tcaccagccc ctgttctcgt cggcaaaaga gccgttcatt tcaataaacc gggcgacctc
     3121 agccatccct tcctgatttt ccgctttcca gcgttcggca cgcagacgac gggcttcatt
     3181 ctgcatggtt gtgcttaccg aaccggagat attgacatca tatatgcctt gagcaactga
     3241 tagctgtcgc tgtcaactgt cactgtaata cgctgcttca tagcatacct ctttttgaca
     3301 tacttcgggt atacatatca gtatatattc ttataccgca aaaatcagcg cgcaaatacg
     3361 catactgtta tctggctttt agtaagccgg atcctctaga ttacgccccg cctgccactc
     3421 atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc
     3481 atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc
     3541 catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt
     3601 gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa
     3661 ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg
     3721 gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac
     3781 ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg
     3841 gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
     3901 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt
     3961 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga
     4021 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
     4081 aaatctcgac ggatcctaac tcaaaatcca cacattatac gagccggaag cataaagtgt
     4141 aaagcctggg gtgcctaatg cggccgccat agtgactgga tatgttgtgt tttacagtat
     4201 tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac
     4261 gtttctcgtt caactttatt atacatagtt gataattcac tggccgtcgt tttacatggc
     4321 acgcccttct attacagggt ttctgataac aaagggtgcc ggatctgcgg cttactcacc
     4381 agacctggcc ccatccgatt accacctatt cgcttcgatg ggacacgcac tcgctgagca
     4441 gcgcttcgat tcttacgaaa gtgtgaaaaa atggctcgat gaatggttcg ccgcaaaaga
     4501 cgatgagttc tactggcgtg gaatccacaa attgcccgag agatgggaaa aatgtgtagc
     4561 tagcgacggc aaatactttg aataaatgat tttttctttt tccacaaaat ttaacgtgtt
     4621 ttttgattta aaaaaaacga catttcatac ttgtacacct gatatctaga tacctaggtg
     4681 agctctggta cccccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     4741 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     4801 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4861 accgcatatg gtgcactctc agtgcaatct gctctgatgc cgcatagtta agccagcccc
     4921 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4981 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     5041 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     5101 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5161 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5221 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5281 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5341 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5401 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5461 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5521 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5581 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5641 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5701 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5761 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5821 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5881 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     5941 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggtcag
     6001 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6061 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6121 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6181 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6241 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6301 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6361 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6421 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6481 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6541 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6601 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6661 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6721 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6781 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6841 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6901 tcctggtctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cc
//
