LOCUS       sequence-370685-        6668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite Gateway [4-3] Destination vector; lox2272-flanked 
            Cbr-unc-119 marker; homology to 5' miniMos Arm.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6668)
  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 6668)
  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: pMLS715
FEATURES             Location/Qualifiers
     source          1..6668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     201..218
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     202..218
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    226..349
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(374..379,380..397,398..404)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             458..1138
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(524..543)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1458..1763
                     /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     1642..1661
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1804..1927)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    2082..2115
                     /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             4075..4086
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    4235..4268
                     /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     4739..4761
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4799..4817)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4885..4989
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4990..5058,5059..5850)
                     /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(5208..5227)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6021..6609
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6510..6529
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
       61 tgataccagc ggataacaat ttcacatcag gtgtacaagt atgaaatgtc gtttcgaaca
      121 tatagatgtc tcgcaaacgt aaatatttat cgattgtcat aaaactttga ccttgtgaag
      181 tgtcaacctt gactgtcgaa tgtaaaacga cggccagtga attatcaact atgtataata
      241 aagttgaacg agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata
      301 aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatgg cggccgcatt
      361 aggcacccca ggctttacac tttatgcttc cggctcgtat aatgtgtgga ttttgagtta
      421 ggatccgtcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
      481 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
      541 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
      601 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
      661 tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
      721 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
      781 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
      841 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
      901 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
      961 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
     1021 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
     1081 acaacagtac tgcgatgagt ggcaggcggg gcgtaatcta gaggatccgg cttactaaaa
     1141 gccagataac agtatgcgta tttgcgcgct gatttttgcg gtataagaat atatactgat
     1201 atgtataccc gaagtatgtc aaaaagaggt atgctatgaa gcagcgtatt acagtgacag
     1261 ttgacagcga cagctatcag ttgctcaagg catatatgat gtcaatatct ccggttcggt
     1321 aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa
     1381 tcaggaaggg atggctgagg tcgcccggtt tattgaaatg aacggctctt ttgccgacga
     1441 gaacaggggc tggtgaaatg cagtttaagg tttacaccta taaaagagag agccgttatc
     1501 gtctgtttgt ggatgtacag agtgatatta ttgacacgcc cgggcgacgg atggtgatcc
     1561 ccctggccag tgcacgtctg ctgtcagata aagtcccccg tgaactttac ccggtggtgc
     1621 atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg
     1681 ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta
     1741 acctgatgtt ctggggaata taaatgtcag gctcccttat acacagccag tctgcaggtc
     1801 gaccatagtg actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa
     1861 atctaattta atatattgat atttatatca ttttacgttt ctcgttcaac ttttctatac
     1921 aaagttgata gcttggcgta atcatggtca tagctgttcc tgcctaggtg agctctggta
     1981 ccctctagtc aaggccttaa gtgagtcgta ttacgtagcc cgggcctagt tctagtttaa
     2041 accacgtgcg tgagcgtaag tttaaacatg attttactaa cataacttcg tataaagtat
     2101 cctatacgaa gttatgacat tctctaatga aaaaatcttt cagttgaaat tgaaaatgag
     2161 ttaaagttgg agtttttatt gaaaacagat ttccgtgtga ttagtgtttt tagcgagtgt
     2221 gacaggacag cgaaaaaata tagaaacaag gggggaactg aaaagcttag gaatgcattg
     2281 aacatgagaa ggggaaaggg aaggaacaaa ctagacagga attattggaa tttaatcaca
     2341 tttggagttt ttttttctat tcgacagaat aattatccag aacatttttg tattaaatat
     2401 ttatgcatca tatgagtagt cggctttgtt gtgcatgacg agtttgttat cgacgaaata
     2461 gaagctgtca gaacgagtct cgtttggatt gttgatcatg tcgtccactg aaaaagagat
     2521 tagtcttttg aattgtactt tttagaataa tgactcactg agttgttgag agagttgagg
     2581 gaactcatag atatgttcac agttgtttcg tgaattcgga atacagaatc cgaattcaaa
     2641 gtcaaaacac ttcagaaggc gatctttgaa gaagtgacgt tcgatcattc ggaaatgatg
     2701 gattgggatg tctcctactt tgaattccac agttgctccg accgttttga gtttcaaaaa
     2761 gtttggagca aatctataac gcacgtatct tgccgactcc tgtggcgact catcattctc
     2821 ctgatcgttc tccggtttgg cgatctcgaa aagcacttgc tcagtgtcca gatcacggat
     2881 ttggaacttg gtgaactcga tgttatagat gttcgcagat ggggagcata agaatcctaa
     2941 atttatgttt taaactgaaa tccaaaggga gcaagatacc ttgagtgatt cccggaagtg
     3001 ctaaaacgtc gttcggagtg atttgagctt tcttcgcaag ctccgattcc gttgtgattc
     3061 cttgttcggt gcttggtggt ggccgtggca tctggaaata tggaaaagtt caacaaaaag
     3121 aaaagagaaa agaatgaaat cggatatcaa gagttagttg agcggtttct ctagttttct
     3181 gagtctcacc tgcgacggga aggtcgccga gccgggtgga atcgatcgtt gttgctcggc
     3241 tttcatatcg gtttggttgg aagcggctga aaacggaaag aagtggaaga aggaaaagag
     3301 tgtggtgtga caggaaaatg gtaattagag ggtgccaaat aaccagctat attttgtttt
     3361 tttttgaaaa catttttaaa aagaaaaata cgataatgat atcagatgga tttccggaaa
     3421 actggtatga aaaatttcaa cctttttgag tacatgtaat caaaatacac tttgtaaatt
     3481 atcattttta ttgaaactcc accatttttc tatttataac gctaataatt tgaaaaagaa
     3541 acctattgcg aaccgcgggg tgaatcccaa aaacgaatgc gttttggtgg agtgattgat
     3601 tcgaatcgaa gaagaaaaag aagaagacgt ggaatagaga gctcactctt aaccgagcag
     3661 cacacaccga cagaaaaaaa aatgaaatga atgagggtct tcttcttctt cttcttcgaa
     3721 tgattgacag aaatgggaaa aagaggaaga ttgagaaggg aaaaaggaag gagaaaagaa
     3781 gcagaagaag acgtcagaga ggagaggaac gagcggaaaa gcagcgggcg caagtcatag
     3841 aagtagcaga gctggggaga agaagacact atccaagaaa ggaatgacga gagagtatgc
     3901 aaaggggtat agggtgcaga cagaatagga acagaataac agatgatgag ccaagaagag
     3961 ttgaaaaggg cgatgaattt gtcatgtaac ttaatttggg tcaatttgag catgatgaat
     4021 tgaaatcatc ccttgttggg agttaataac cggtttgtta tcagaaaccc tgtaatagaa
     4081 gggcgcccta actttgagcc aattcatccc ggtttctgtc aaatatatca aaaagtggtc
     4141 aactgacaaa ttgtttttga tattataata aacattttat ccgttaacaa ttttcgaata
     4201 ctttttacaa ggacttggat aaattggctc aaagataact tcgtataaag tatcctatac
     4261 gaagttatta actaatctga tttaaatttt cgcgaattga gcgtcataat tgtttactct
     4321 cagtgcagtc aacatgtcga gtttcgtgcc gaataaagag caaacgcgga cagtattaat
     4381 tttctgtttt catttgaaga aaacagctgc ggacgtcgtg actgggaaaa ccctggcgtt
     4441 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     4501 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg
     4561 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
     4621 gcaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     4681 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4741 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     4801 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     4861 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     4921 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     4981 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5041 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5101 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5161 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5221 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5281 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5341 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5401 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5461 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5521 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5581 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5641 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     5701 cgtgggtctc gcggtatcat tgcagcactg gggtcagatg gtaagccctc ccgtatcgta
     5761 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     5821 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     5881 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     5941 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6001 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6061 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6121 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6181 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6241 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6301 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6361 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6421 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6481 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6541 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6601 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggtcttttg ctggcctttt
     6661 gctcacat
//
