LOCUS       40924_45398        8382 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pUCTCAPR2-GW, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8382)
  AUTHORS   Kvitko BH, McMillan IA, Schweizer HP.
  TITLE     An Improved Method for oriT-Directed Cloning and Functionalization 
            of Large Bacterial Genomic Regions
  JOURNAL   Appl. Environ. Microbiol. 79 (16), 4869-4878 (2013)
  PUBMED    23747708
REFERENCE   2  (bases 1 to 8382)
  AUTHORS   Kvitko BH, McMillan IA, Schweizer HP.
  TITLE     Direct Submission
  JOURNAL   Submitted (03-SEP-2012) Microbiology Immunology and Pathology, 
            Colorado State University, IDRC at Foothills Campus, Campus Delivery
            0922, Fort Collins, CO 80523-0922, USA
REFERENCE   3  (bases 1 to 8382)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8382)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Appl. 
            Environ. Microbiol."; date: "2013"; volume: "79"; issue: "16"; 
            pages: "4869-4878"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (03-SEP-2012) Microbiology Immunology and Pathology, Colorado State 
            University, IDRC at Foothills Campus, Campus Delivery 0922, Fort 
            Collins, CO 80523-0922, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8382
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..372
                     /codon_start=1
                     /label=BleoR
                     /note="antibiotic-binding protein"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     misc_recomb     486..533
                     /label=FRT
                     /note="FRT"
     protein_bind    complement(486..533)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     terminator      complement(586..672)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      complement(775..869)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(897..913)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   918..1116
                     /label=Tn7R
                     /note="Tn7R"
     misc_feature    1264..1281
                     /label=I-SceI restriction site
                     /note="I-SceI restriction site"
     primer_bind     complement(1291..1307)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      1657..1743
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      1835..1862
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     oriT            2309..2418
                     /label=oriT
                     /note="incP origin of transfer"
     terminator      complement(3117..3160)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      3292..3319
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        3338..3429
                     /label=AmpR promoter
     CDS             complement(3895..4425)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(4614..4642)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      5365..5953
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    6241..6262
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        6277..6307
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6315..6331
                     /label=lac operator
                     /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     6339..6355
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    6374..6391
                     /label=I-SceI restriction site
                     /note="I-SceI restriction site"
     protein_bind    6414..6538
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        6563..6593
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             6647..7303
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             7648..7950
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(7994..8118)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(8180..8227)
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        8317..8364
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
ORIGIN
        1 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc
       61 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt
      121 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac
      181 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag
      241 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag
      301 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc
      361 gaggagcagg actgatcatg atgatatatt tttatcttgt gcaatgtaac atcagagata
      421 agccgaatta tctagaatta ttccattgag taagttttta agcacatcag cttcaaaagc
      481 gctctgaagt tcctatactt tctagagaat aggaacttcg gaataggtac ttcaagatcc
      541 ccaattcgag ctcgattcgt ccgggccgca agctcctagc ggcggatttg tcctactcag
      601 gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt
      661 tcgttttatt tgatgcctca agctagagag tcattacccc aggcgtttaa gggcaccaat
      721 aactgcctta aaaaaattac gccccgccct gccactcatc gcagtctagc ttggattctc
      781 accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga
      841 ggtcattact ggatctatca acaggagtcc aagctcagct aattaagcta gcttatcgat
      901 accgtcgacc tcgaacccca cgcccctctt taatacgacg ggcaatttgc acttcagaaa
      961 atgaagagtt tgctttagcc ataacaaaag tccagtatgc tttttcacag cataactgga
     1021 ctgatttcag tttacaacta ttctgtctag tttaagactt tattgtcata gtttagatct
     1081 attttgttca gtttaagact ttattgtccg cccacacccg cttacgcagg gcatccattt
     1141 attactcaac cgtaaccgat tttgccaggt tacgcggctg gtcctctagc tggcgtaata
     1201 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     1261 gggtagggat aacagggtaa tgagcttggc actggccgtc gttttacaac gtcgtgactg
     1321 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     1381 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     1441 cgaatggcga gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta
     1501 aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg
     1561 tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg
     1621 ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg
     1681 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     1741 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga
     1801 cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt
     1861 tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata tgcatgcgcc
     1921 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tcgacatccg
     1981 ccctcaccgc caggaacgca accgcagcct catcacgccg gcgcttcttg gccgcgcggg
     2041 attcaaccca ctcggccagc tcgtcggtgt agctctttgg catcgtctct cgcctgtccc
     2101 ctcagttcag taatttcctg catttgcctg tttccagtcg gtagatattc cacaaaacag
     2161 cagggaagca gcgcttttcc gctgcataac cctgcttcgg ggtcattata gcgatttttt
     2221 cggtatatcc atcctttttc gcacgatata caggattttg ccaaagggtt cgtgtagact
     2281 ttccttggtg tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga
     2341 gcgggtgttc cttcttcact gtcccttatt cgcacctggc ggtgctcaac gggaatcctg
     2401 ctctgcgagg ctggccggct accgccggcg taacagatga gggcaagcgg atggctgatg
     2461 aaaccaagcc aaccaggaag ggcagcccac ctatcaaggt gtactgcctt ccagacgaac
     2521 gaagagcgat tgaggaaaag gcggcggcgg ccggcatgag cctgtcggcc tacctgctgg
     2581 ccgtcggcca gggctacaaa atcacgggcg tcgtggacta tgagcacgtc cgcgagctgg
     2641 cccgcatcaa tggcgacctg ggccgcctgg gcggcctgct gaaactctgg ctcaccgacg
     2701 acccgcgcac ggcgcggttc ggtgatgcca cgatcctcgc cctgctggcg aagatcgact
     2761 ctagctagag gatcgattct atgtcttacc tgatcttcta caacctgatc aaaccgtacc
     2821 tgatcccgca gatgatgtac aaactgccga acactatctc ctccgaaact ttcctgaaat
     2881 aagggcccga cgtctgcagg catgcaagct tggctgtttt ggcggatgag agaagatttt
     2941 cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg
     3001 cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag
     3061 cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa
     3121 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     3181 ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg
     3241 gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca
     3301 tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt ctaaatacat
     3361 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3421 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3481 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3541 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3601 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3661 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3721 aatgacttgg ttgagtaagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3781 atttaacaaa aatttaacgc gaattttaac aaaatcgaat tggccgcggc gttgtgacaa
     3841 tttaccgaac aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc
     3901 ggtacttggg tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag
     3961 agccactgcg ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc
     4021 gttggcctca tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc
     4081 gccggagact gcgagatcat agatatagat ctcactacgc ggctgctcaa acctgggcag
     4141 aacgtaagcc gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa
     4201 tgtcttacta cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt
     4261 ggctacgtct ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg
     4321 gtcagggccg agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt
     4381 agggcgactg ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata
     4441 acatcaaaca tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact
     4501 gtacaaaaaa acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc
     4561 gttcggtcaa ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac
     4621 gaaccgaaca ggcttatgtc aattcgatta ttgaagcatt tatcagggtt attgtctcat
     4681 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4741 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     4801 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct
     4861 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     4921 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc
     4981 ggcatcagag cagattgtac tgagagtgca ccataatcgg cattttcttt tgcgttttta
     5041 tttgtttggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     5101 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     5161 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     5221 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5281 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5341 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5401 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5461 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     5521 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5581 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5641 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5701 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5761 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5821 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5881 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5941 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6001 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     6061 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     6121 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     6181 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     6241 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     6301 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     6361 ttacgccaag ctctagggat aacagggtaa tcccccttcg cgaggtaccc atcacaagtt
     6421 tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga
     6481 ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag tcactatggc
     6541 ggccgcatta ggcaccccag gctttacact ttatgcttcc ggctcgtata atgtgtggat
     6601 tttgagttag ggtccggcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat
     6661 cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt
     6721 tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt
     6781 aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg
     6841 cctgatgaat gctcatccgg agttccgtat ggcaatgaaa gacggtgagc tggtgatatg
     6901 ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct
     6961 ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc
     7021 gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt
     7081 ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa
     7141 cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat
     7201 gccgctggcg attcaggttc atcatgccgt ctgtgatggc ttccatgtcg gcagaatgct
     7261 taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaaacgc gtgggtccgg
     7321 cttactaaaa gccagataac agtatgcgta tttgcgcgct gatttttgcg gtataagaat
     7381 atatactgat atgtataccc gaagtatgtc aaaaagaggt gtgctatgaa gcagcgtatt
     7441 acagtgacag ttgacagcga cagctatcag ttgctcaagg catatatgat gtcaatatct
     7501 ccggtctggt aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga
     7561 aagcggaaaa tcaggaaggg atggctgagg tcgcccggtt tattgaaatg aacggctctt
     7621 ttgctgacga gaacagggac tggtgaaatg cagtttaagg tttacaccta taaaagagag
     7681 agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc cgggcgacgg
     7741 atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac
     7801 ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg
     7861 ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa
     7921 aacgccatta acctgatgtt ctggggaata taaatgtcag gctcccttat acacagccag
     7981 tctgcaggtc gaccatagtg actggatatg ttgtgtttta cagtattatg tagtctgttt
     8041 tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt ctcgttcagc
     8101 tttcttgtac aaagtggtga tggggatcca ctagtgagct catgcatgat cgagctcgaa
     8161 ttagcttcaa aagcgctctg aagttcctat actttctaga gaataggaac ttcggaatag
     8221 gaacttcaag atcccctgat tccctttgtc aacagcaatg gatttcggct tcgacgcgta
     8281 actcacgtta agggattttg gtcatgatca gcacgtgttg acaattaatc atcggcatag
     8341 tatatcggca tagtataata cgacaaggtg aggaactaaa cc
//
