LOCUS       sequence-222772-        4438 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 4438)
  TITLE     Grefen Lab pUC-L Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4438)
  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            "Unpublished"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-L3L2
FEATURES             Location/Qualifiers
     source          1..4438
                     /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    218..313
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        463..565
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             566..1249
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(632..651)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1569..1874
                     /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     1753..1772
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1916..2015)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2033..2049)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2033..2049)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2046..2068)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2057..2073
                     /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(2081..2087,2088..2105,2106..2111))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2126..2147
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2264..2281)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2435..3023)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2515..2534)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3194..3985,3986..4054))
                     /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     3817..3836
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4055..4159)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4227..4245
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4283..4305)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4405..4424
                     /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 tataataaag ttgctagcgt cgactaagtt ggcagcatca cccgacgcac tttgcgccga
      361 ataaatacct gtgacggaag atcacttcgc agaataaata aatcctggtg tccctgttga
      421 taccgggaag ccctgggcca acttttggcg aaaatgagac gttgatcggc acgtaagagg
      481 ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg agttatcgag
      541 attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga
      601 tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac
      661 ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa
      721 gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga
      781 attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta
      841 caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga
      901 tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc
      961 ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag
     1021 tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac
     1081 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
     1141 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
     1201 cgatgagtgg cagggggggc gtaaacgccg cgtggatccg gcttactaaa agccagataa
     1261 cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa tatatactga tatgtatacc
     1321 cgaagtatgt caaaaagagg tatgctatga agcagcgtat tacagtgaca gttgacagcg
     1381 acagctatca gttgctcaag gcatatatga tgtcaatatc tccggtctgg taagcacaac
     1441 catgcagaat gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg
     1501 gatggctgag gtcgcccggt ttattgaaat gaacggctct tttgctgacg agaacagggg
     1561 ctggtgaaat gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg
     1621 tggatgtaca gagtgatatt attgacacgc ccgggcgacg gatggtgatc cccctggcca
     1681 gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg
     1741 atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg
     1801 aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt
     1861 tctggggaat ataaatgtca ggctccctta tacacagcca gtctgcaggc cggctaccca
     1921 gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa
     1981 caggtcacta tcagtcaaaa taaaatcatt atttaagctt ggcgtaatca tggtcatagc
     2041 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     2101 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     2161 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     2221 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2281 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2341 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2401 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2461 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2521 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2581 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2641 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2701 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2761 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2821 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     2881 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2941 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3001 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3061 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3121 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3181 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3241 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3301 taccatctgg ccccagtgct gcaatgatac cgcgagatcc acgctcaccg gctccagatt
     3361 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3421 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3481 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3541 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3601 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3661 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3721 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3781 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3841 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3901 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     3961 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4021 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4081 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4141 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     4201 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttgcgc
     4261 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagact gtcacagctt
     4321 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     4381 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcacca
//
