LOCUS       sequence-356074-        7373 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Plasmid for unmarked deletions, pUC19 backbone, Apr resistant 
            casette, codA::app counterselection casette, gateway compatible.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7373)
  AUTHORS   Stevens DM, Tang A, Coaker G
  TITLE     A Genetic Toolkit for Investigating Clavibacter Species: Markerless 
            Deletion, Permissive Site Identification, and an Integrative 
            Plasmid.
  JOURNAL   Mol Plant Microbe Interact. 2021 Dec;34(12):1336-1345. doi: 
            10.1094/MPMI-07-21-0171-TA. Epub 2021 Dec 10.
  PUBMED    34890250
REFERENCE   2  (bases 1 to 7373)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1094/MPMI-07-21-0171-TA"; journalName: "Mol Plant Microbe 
            Interact"; date: "2021-12"; volume: "34"; issue: "12"; pages: 
            "1336-1345"
COMMENT     Sequence Label: pSelAct-KO
FEATURES             Location/Qualifiers
     source          1..7373
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     35..51
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     35..51
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    61..185
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(210..215,216..233,234..240)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             294..974
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(360..379)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1294..1599
                     /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     1478..1497
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1640..1764)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1799..1815)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1845..1865)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1845..1863)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1884..1900)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1884..1900)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1897..1919)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1908..1924
                     /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(1932..1938,1939..1956,1957..1962))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1977..1998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2115..2132)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2286..2874)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2366..2385)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2991..3791)
                     /codon_start=1
                     /gene="aac(3)IV"
                     /product="aminoglycoside 3-N-acetyltransferase type IV     
                               "
                     /label=ApmR
                     /note="confers resistance to apramycin (Paget and Davies, 
                     1996)"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
     oriT            4107..4216
                     /note="incP origin of transfer"
     regulatory      4791..4800
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4797..6080
                     /codon_start=1
                     /gene="codA"
                     /product="E. coli cytosine deaminase"
                     /label=codA
                     /note="confers sensitivity to 5-fluorocytosine"
                     /translation="MVSNNALETIINARLPGEEGLWQIHLQDGKISAIDAQSGVMPITE
                     NSLDAEQGLVIPPFVEPHIHLDTTQTAGQPNWNQSGTLFEGIERWAERKALLTHDDVKQ
                     RAWQTLKWQIANGIQHVRTHVDVSDATLTALKAMLEVKQEVAPWIDLQIVAFPQEGILS
                     YPNGEALLEEALRLGADVVGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDD
                     EQSRFVETVAALAHHEGMGARVTASHTTAMHSYNGAYTSRLFRLLKMSGINFVANPLVN
                     IHLQGRFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVC
                     QLMGYGQINDGLNLITHHSARTLNLQDYGIAAGNSANLIILPAENGFDALRRQVPVRYS
                     VRGGKVIASTQPAQTTVYLEQPEAIDYKR"
     rep_origin      complement(6761..7189)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6871..6892)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7083..7102
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7316..7338
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7330..7347
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7331..7347
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(7357..7373,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(7357..7373,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg gagctccacc gcggtggcgg ccgctctaga actagtggat cccccatcaa
       61 acaagtttgt acaaaaaagc tgaacgagaa acgtaaaatg atataaatat caatatatta
      121 aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca
      181 ctatggcggc cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg
      241 tgtggatttt gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga
      301 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
      361 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
      421 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
      481 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
      541 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
      601 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
      661 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
      721 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
      781 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
      841 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
      901 gaatgcttaa tgaattacaa cagtactgcg atgagtggca ggcggggcgt aatctagagg
      961 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     1021 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     1081 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     1141 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     1201 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     1261 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     1321 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     1381 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt cccccgtgaa
     1441 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     1501 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     1561 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     1621 agccagtctg caggtcgacc atagtgactg gatatgttgt gttttacagt attatgtagt
     1681 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
     1741 ttcagctttc ttgtacaaag tggttcgatg ggctgcagga attcgatatc aagcttatcg
     1801 ataccgtcga cctcgagggg gggcccggta cccagctttt gttcccttta gtgagggtta
     1861 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     1921 acaattccac acaacatacg agccgggagc ataaagtgta aagcctgggg tgcctaatga
     1981 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     2041 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     2101 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2161 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2221 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2281 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2341 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2401 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2461 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2521 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     2581 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2641 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2701 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2761 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2821 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2881 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2941 ttggtcatga ctagagaata ggaacttcgg aataggaact tatgagctca gccaatcgac
     3001 tggcgagcgg catcgcattc ttcgcatccc gcctctggcg gatgcaggaa gatcaacgga
     3061 tctcggccca gttgacccag ggctgtcgcc acaatgtcgc gggagcggat caaccgagca
     3121 aaggcatgac cgactggacc ttccttctga aggctcttct ccttgagcca cctgtccgcc
     3181 aaggcaaagc gctcacagca gtggtcattc tcgagataat cgacgcgtac caacttgcca
     3241 tcctgaagaa tggtgcagtg tctcggcacc ccatagggaa cctttgccat caactcggca
     3301 agatgcagcg tcgtgttggc atcgtgtccc acgccgagga gaagtacctg cccatcgagt
     3361 tcatggacac gggcgaccgg gcttgcaggc gagtgaggtg gcaggggcaa tggatcagag
     3421 atgatctgct ctgcctgtgg ccccgctgcc gcaaaggcaa atggatgggc gctgcgcttt
     3481 acatttggca ggcgccagaa tgtgtcagag acaactccaa ggtccggtgt aacgggcgac
     3541 gtggcaggat cgaacggctc gtcgtccaga cctgaccacg agggcatgac gagcgtccct
     3601 cccggaccca gcgcagcacg cagggcctcg atcagtccaa gtggcccatc ttcgaggggc
     3661 cggacgctac ggaaggagct gtggaccagc agcacaccgc cgggggtaac cccaaggttg
     3721 agaagctgac cgatgagctc ggcttttcgc cattcgtatt gcacgacatt gcactccacc
     3781 gctgatgaca tcagtcgatc atagcacgat caacggcact gttgcaaata gtcggtggtg
     3841 ataaacttat catccccttt tgcttatgga gctgcacatg aacccattca aaggccggca
     3901 ttttcagcgt gacatcattc tgtgggccgt acgctggtac tgcaaatacg gcatcagtta
     3961 ccgtgagctg cattttccgc tgcataaccc tgcttcgggg tcattatagc gattttttcg
     4021 gtatatccat cctttttcgc acgatataca ggattttgcc aaagggttcg tgtagacttt
     4081 ccttggtgta tccaacggcg tcagccgggc aggataggtg aagtaggccc acccgcgagc
     4141 gggtgttcct tcttcactgt cccttattcg cacctggcgg tgctcaacgg gaatcctgct
     4201 ctgcgaggct ggcgggaact tcgaagttcc tatactttct aggcatgagc ggatacatat
     4261 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4321 cacctaaatt gtaagcgtta ataattcgat agcttcacgc tgccgcaagc actcagggcg
     4381 caagggctgc taaaggaagc ggaacacgta gaaagccagt ccgcagaaac ggtgctgacc
     4441 ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa
     4501 gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt tatggacagc
     4561 aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gtttgggaag ccctgcaaag
     4621 taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca agatcgatct
     4681 gatcaagaga caggatgagg atcgtttcgc atgatatcat caagcttatc gataccgtcg
     4741 actactaacc ttcttctctt tcctacagct gagatcaccg gcgaaggagg gccaccatgg
     4801 tgtcgaataa cgctttagaa acaattatta acgcccggtt accaggcgaa gaggggctgt
     4861 ggcagattca tctgcaggac ggaaaaatca gcgccattga tgcgcaatcc ggcgtgatgc
     4921 ccataactga aaacagcctg gatgccgaac aaggtttagt tataccgccg tttgtggagc
     4981 cacatattca cctggacacc acgcaaaccg ccggacaacc gaactggaat cagtccggca
     5041 cgctgtttga aggcattgaa cgctgggccg agcgcaaagc gttattaacc catgacgatg
     5101 tgaaacaacg cgcatggcaa acgctgaaat ggcagattgc caacggcatt cagcatgtgc
     5161 gtacccatgt cgatgtttcg gatgcaacgc taactgcgct gaaagcaatg ctggaagtga
     5221 agcaggaagt cgcgccgtgg attgatctgc aaatcgtcgc cttccctcag gaagggattt
     5281 tgtcgtatcc caacggtgaa gcgttgctgg aagaggcgtt acgcttaggg gcagatgtag
     5341 tgggggcgat tccgcatttt gaatttaccc gtgaatacgg cgtggagtcg ctgcataaaa
     5401 ccttcgccct ggcgcaaaaa tacgaccgtc tcatcgacgt tcactgtgat gagatcgatg
     5461 acgagcagtc gcgctttgtc gaaaccgttg ctgccctggc gcaccatgaa ggcatgggcg
     5521 cgcgagtcac cgccagccac accacggcaa tgcactccta taacggggcg tatacctcac
     5581 gcctgttccg cttgctgaaa atgtccggta ttaactttgt cgccaacccg ctggtcaata
     5641 ttcatctgca aggacgtttc gatacgtatc caaaacgtcg cggcatcacg cgcgttaaag
     5701 agatgctgga gtccggcatt aacgtctgct ttggtcacga tgatgtcttc gatccgtggt
     5761 atccgctggg aacggcgaat atgctgcaag tgctgcatat ggggctgcat gtttgccagt
     5821 tgatgggcta cgggcagatt aacgatggcc tgaatttaat cacccaccac agcgcaagga
     5881 cgttgaattt gcaggattac ggcattgccg ccggaaacag cgccaacctg attatcctgc
     5941 cggctgaaaa tgggtttgat gcgctgcgcc gtcaggttcc ggtacgttat tcggtacgtg
     6001 gcggcaaggt gattgccagc acacaaccgg cacaaaccac cgtatatctg gagcagccag
     6061 aagccatcga ttacaaacgt atcagcggag ctaatggcgt catggctaag atcgtggaag
     6121 tcaaacaccc actcgtcaaa cacaagctgg gactgatgcg tgagcaagat atcagcacca
     6181 agcgctttcg cgaactcgct tccgaagtgg gtagcctgct gacttacgaa gcgaccgccg
     6241 acctcgaaac ggaaaaagta actatcgaag gctggaacgg cccggtagaa atcgaccaga
     6301 tcaaaggtaa gaaaattacc gttgtgccaa ttctgcgtgc gggtcttggt atgatggacg
     6361 gtgtgctgga aaacgttccg agcgcgcgca tcagcgttgt cggtatgtac cgtaatgaag
     6421 aaacgctgga gccggtaccg tacttccaga aactggtttc taacatcgat gagcgtatgg
     6481 cgctgatcgt tgacccaatg ctggcaaccg gtggttccgt tatcgcgacc atcgacctgc
     6541 tgaaaaaagc gggctgcagc agcatcaaag ttctggtgct ggtagctgcg ccagaaggta
     6601 tcgctgcgct ggaaaaagcg cacccggacg tcgaactgta taccgcatcg attgatcagg
     6661 gactgaacga gcacggatac attattccgg gcctcggcga tgccggtgac aaaatctttg
     6721 gtacgaaata agcttgtcga aattcgctag attttgttaa aattcgcgtt aaatttttgt
     6781 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     6841 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     6901 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     6961 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     7021 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     7081 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     7141 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
     7201 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     7261 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     7321 tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact ata
//
