LOCUS       sequence-117439-        5347 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini Tol2 backbone with att4-attR2 cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5347)
  TITLE     Other Lawson Lab plasmids
REFERENCE   2  (bases 1 to 5347)
  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"
COMMENT     Sequence Label: pminTolR4-R2
FEATURES             Location/Qualifiers
     source          1..5347
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..41)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(24..40)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(33..55)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      181..636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     478..499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        714..818
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(819..887,888..1679)
                     /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(1037..1056)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1850..2438
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2339..2358
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2592..2609
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2726..2747
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2762..2767,2768..2785,2786..2792)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2800..2816
                     /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     2805..2827
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2824..2840
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2824..2840
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2858..2876
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2858..2875
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    join(2927..2938,2939..3076)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    3109..3232
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3257..3262,3263..3280,3281..3287)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3341..4000
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3407..3426)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4342..4647
                     /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     4526..4545
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4688..4811)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    4828..4962
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4852..4871)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4906..4925
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(5134..5321,5322..5333)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(join(5345..5347,1..17))
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(join(5346..5347,1..17))
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
       61 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
      121 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
      181 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
      241 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      301 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      361 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      421 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      481 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      541 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      601 acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac
      661 gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat gtgcgcggaa
      721 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
      781 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
      841 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
      901 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
      961 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1021 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1081 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1141 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1201 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1261 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1321 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     1381 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1441 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1501 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     1561 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     1621 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     1681 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     1741 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     1801 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1861 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1921 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1981 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2041 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2101 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2161 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2221 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2281 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2341 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2401 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2461 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2521 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2581 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     2641 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     2701 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     2761 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     2821 acacaggaaa cagctatgac catgattacg ccaagctatt taggtgacac tatagaatac
     2881 tcaagctatg catccaacgc gttgggagct ctcccatatg gtcgagcaga ggtgtaaaaa
     2941 gtactcaaaa attttactca agtgaaagta caagtactta gggaaaattt tactcaatta
     3001 aaagtaaaag tatctggcta gaatcttact tgagtaaaag taaaaaagta ctccattaaa
     3061 attgtacttg agtattaagg aagtagggat aacagggtaa tctcgaatca actttgtata
     3121 gaaaagttga acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc
     3181 ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta tggcggccgc
     3241 attaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt ggattttgag
     3301 ttaggatccg tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg
     3361 atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc
     3421 agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac
     3481 cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat
     3541 gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag
     3601 tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag
     3661 tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta
     3721 cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc
     3781 caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt
     3841 cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct
     3901 ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga
     3961 attacaacag tactgcgatg agtggcaggg cggggcgtaa tctagaggat ccggcttact
     4021 aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa gaatatatac
     4081 tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg
     4141 acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat atctccggtt
     4201 cggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg
     4261 aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc tcctttgccg
     4321 acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag agagagccgt
     4381 tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg
     4441 atccccctgg ccagtgcacg tctgctgtca gataaagtcc cccgtgaact ttacccggtg
     4501 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc
     4561 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc
     4621 attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag ccagtctgca
     4681 ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct gttttttatg
     4741 caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt caactttctt
     4801 gtacaaagtt gatgtaccat cgatgatgat ccagacatga taagatacat tgatgagttt
     4861 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     4921 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     4981 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
     5041 tacaaatgtg gtatggctga ttatgatcct ctagatcgtg catgcttccg cggttagatc
     5101 tattaccctg ttatccctag ggaaaataga atgaagtgat ctccaaaaaa taagtacttt
     5161 ttgactgtaa ataaaattgt aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt
     5221 aagtaaaagt aaaagtattg atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa
     5281 taatacttaa gtacagtaat caagtaaaat tactcaagta ctttacacct ctgggcccaa
     5341 ttcgccc
//
