LOCUS       sequence-222765-        4441 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Create Entry clones for Gateway LR reactions through simple 
            restriction enzyme based cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4441)
  TITLE     Grefen Lab pUC-L Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4441)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pUC-L1L4
FEATURES             Location/Qualifiers
     source          1..4441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     180..202
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     194..211
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     195..211
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    217..316
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        466..568
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             569..1252
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(635..654)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1572..1877
                     /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     1756..1775
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1922..2017)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2036..2052)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2036..2052)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2049..2071)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2060..2076
                     /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)."
     promoter        complement(join(2084..2090,2091..2108,2109..2114))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2129..2150
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2267..2284)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2438..3026)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2518..2537)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3197..3988,3989..4057))
                     /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     3820..3839
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4058..4162)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4230..4248
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4286..4308)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4408..4427
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc
       61 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
      121 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
      181 ccagtcacga cgttgtaaaa cgacggccag tgaattcaaa taatgatttt attttgactg
      241 atagtgacct gttcgttgca acaaattgat gagcaatgct tttttataat gccaactttg
      301 tacaaaaaag caggctctag agtcgactaa gttggcagca tcacccgacg cactttgcgc
      361 cgaataaata cctgtgacgg aagatcactt cgcagaataa ataaatcctg gtgtccctgt
      421 tgataccggg aagccctggg ccaacttttg gcgaaaatga gacgttgatc ggcacgtaag
      481 aggttccaac tttcaccata atgaaataag atcactaccg ggcgtatttt ttgagttatc
      541 gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt
      601 tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg
      661 tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa
      721 taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc
      781 ggaattccgt atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg
      841 ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga
      901 cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct
      961 ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt
     1021 gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt
     1081 caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt
     1141 tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta
     1201 ctgcgatgag tggcaggggg ggcgtaaacg ccgcgtggat ccggcttact aaaagccaga
     1261 taacagtatg cgtatttgcg cgctgatttt tgcggtataa gaatatatac tgatatgtat
     1321 acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg acagttgaca
     1381 gcgacagcta tcagttgctc aaggcatata tgatgtcaat atctccggtc tggtaagcac
     1441 aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga
     1501 agggatggct gaggtcgccc ggtttattga aatgaacggc tcttttgctg acgagaacag
     1561 gggctggtga aatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt
     1621 ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg
     1681 ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg
     1741 gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg
     1801 gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga
     1861 tgttctgggg aatataaatg tcaggctccc ttatacacag ccagtctgca ggccggccac
     1921 ccaacttttc tatacaaagt tggcattata agaaagcatt gcttatcaat ttgttgcaac
     1981 gaacaggtca ctatcagtca aaataaaatc attatttaag cttggcgtaa tcatggtcat
     2041 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     2101 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     2161 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2221 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2281 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2341 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2401 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2461 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2521 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2581 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2641 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2701 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2761 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2821 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     2881 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2941 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3001 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3061 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3121 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3181 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3241 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3301 gcttaccatc tggccccagt gctgcaatga taccgcgaga tccacgctca ccggctccag
     3361 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3421 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3481 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3541 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3601 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3661 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3721 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3781 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3841 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3901 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3961 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4021 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4081 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4141 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     4201 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttg
     4261 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag actgtcacag
     4321 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     4381 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     4441 a
//
