LOCUS       Exported                8662 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pKOV
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8662)
  AUTHORS   Link AJ, Phillips D, Church GM
  TITLE     Methods for generating precise deletions and insertions in the 
            genome of wild-type Escherichia coli: application to open reading 
            frame characterization.
  JOURNAL   J Bacteriol. 1997 Oct . 179(20):6228-37.
  PUBMED    9335267
REFERENCE   2  (bases 1 to 8662)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8662
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        118..220
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             221..880
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(287..306)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     3484..3501
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     rep_origin      4111..4565
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4197..4216)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4407..4428
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4669..5114
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             5115..6536
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATVTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     CDS             complement(6929..7879)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      7927..8149
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 ccctttcgtc ttcgaataaa tacctgtgac ggaagatcac ttcgcagaat aaataaatcc
       61 tggtgtccct gttgataccg ggaagccctg ggccaacttt tggcgaaaat gagacgttga
      121 tcggcacgta agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt
      181 ttttgagtta tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg
      241 atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc
      301 agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac
      361 cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat
      421 gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag
      481 tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag
      541 tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta
      601 cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc
      661 caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt
      721 cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct
      781 ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga
      841 attacaacag tactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg
      901 gtgcccttaa acgcctggtt gctacgcctg aataagtgat aataagcgga tgaatggcag
      961 aaattcgaaa gcaaattcga cccggtcgtc ggttcagggc agggtcgtta aatagccgct
     1021 tatgtctatt gctggtttaa tcggtacccg gggatcgcgg ccgccccgaa cccaactgat
     1081 gcagtatccc gggcgctaca acatgatctc agtgatgtta tcggtcagga acaaggaaag
     1141 cgaggactgg aaattaccgc tgctggcggg cacaaccttt tactgattgg gccgccggga
     1201 acaggtaaaa caatgctcgc cagccgtatt aatggcctgt tgccagattt aagcaatgaa
     1261 gaggcactgg agagtgctgc gatattaagt ctggtaaatg ctgaatcagt acaaaaacaa
     1321 tggcggcagc gcccgttccg ctcacctcat cacagtgcat cgttaactgc gatggtaggc
     1381 ggtggcgcaa ttccagggcc cggtgaaatt tcgctggcgc ataacggcgt gctttttctt
     1441 gatgagctac ctgaatttga acggcgtaca ccggatgcct tgcgagagcc gattgaatcc
     1501 gggcagatcc atctttcacg cacacgagca aaaataacct atccagcccg tttccagctt
     1561 gttgcagcga tgaatcccag ccctaccggg cattatcagg gaaaccataa ccgctgcacg
     1621 ccagaacaga cattacgtta tctcaaccgg ctctcggggc cctttctcga ccgcttcgat
     1681 ctctcactgg agatcccatt accacccccc ggcactttga gtaaaacggt agtgccggga
     1741 gaaagcagcg ccaccattaa acaacgcgta atggccgcca gagagcgcca atttaagcgg
     1801 cagaataaac tgaatgcctg gctggatagt ccggaaatac gccaattctg caaacttgag
     1861 agcgaagatg cgatgtggtt ggaaggaaca ctgatccatc tggggttatc gattcgtgcc
     1921 tggcagcggt tattgaaagt tgcacgaacc attgctgata ttgatcagtc tgacattatc
     1981 acacgtcagc atttgcagga ggcagttagc tatcgagcga ttgaccgttt gctcatccat
     2041 ctgcagaaac tactgacata aaaaaagggc atttcgccct ttttattaat cgtcagaatc
     2101 ggtgtagtct tcagcacctt caacctgcgg tttaccgcgg aaagggtgtg aaaacgtttt
     2161 ggacgcttaa tacgcgtcat atacttggac cacacgcgtt ctgcttctgt cactggctca
     2221 cgttcgccac ggcatactgc tacgaagagt ttctcttcct cggtaaccgg ctcgcgtttg
     2281 ccaagatcca actcattgaa ggcataacca tgacgctcaa gcagttgtgc ctctttgatg
     2341 gtgaaatcac catgacgaga gaatccacgt ggataatgtt tattgtcgaa atatcgatta
     2401 gtcgtcgtaa agctttccgc catcctgcac gctcctaatt ctttgaccga gctagttatg
     2461 gcgcggagta ttagttacgc ttgacagagt gtaaaacaaa acatttaaat cataacgaca
     2521 aataattttg cggagagcac tgtggatgtt ataaatttgg agtgtgaagg ttattgcgtg
     2581 tggtaaaaca gctgcatgaa cgccagcttg atcttcttat taccactgaa gcgcccaaaa
     2641 tggacgaatt tagtagtcag ttgctgggat atttcacttt agcgctttat accagtgccc
     2701 cttcaaaact aaagggagat cttaattatc tgcgacttga gtgggggcca gattttcaac
     2761 agcatgaggc aggtttgatc ggtgctgacg aagtgcccat tctgacaacc agttctgctg
     2821 aactggcaca gcaacagatt gcgatgctta atggttgcac ctggctaccc gtcagctggg
     2881 cgcgtaaaaa aggcggcctg cataccgttg tcgatagcac aacactttca cggccgcttt
     2941 atgccatatg gctgcaaaat agcgataaaa atgcgttgat tcgcgatctt ttgaaaatta
     3001 acgtgctgga tgaagtgtat taatatgaat ggctggcaag gatgccggta gaaggattta
     3061 cttcggagag ggttatttca gataaaaaaa atccttacgt ttcgctaagg atgatttctg
     3121 gcaggggcgg agagactcga actcccaaca cccggttttg gagaccggtg ctctaccaat
     3181 tgaactacgc ccctaattag ggtggcggaa cggacgggac tcgaacccgc gaccccctgc
     3241 gtgacaggca ggtattctaa ccgactgaac taccgctcca ccgaattctt ttacaaccac
     3301 cggttttatg accggcttac tgcttaattt gatgcctggc agttccctac tctcgcatgg
     3361 ggagacccca cactaccatc ggcgctacgg cgtttcactt ctgagttcgg catggggtca
     3421 ggtgggacca ccgcgctacg gccgccaggc aaattctgtt tcatcagacc gcttctgcgt
     3481 tctgatttaa tctgtatcag gctgaaaatc ttctctcatc cgccaaaaca tcttcggcgt
     3541 tgtaaggtta agcctcacgg ttcattagta ccggttagct caacgcatcg ctgcgcttac
     3601 acacccggcc tatcaacgtc gtcgtcttca acgttccttc aggactctca aggagtcagg
     3661 gagaactcat ctcggggcaa gtttcgtgct tagatgcttt cagcacttat ctcttccgca
     3721 tttagctacc gggcagtgcc attggcatga caacccgaac accagtgatg cgtccactcc
     3781 ggtcctctcg tactaggagc agcccccctc agttctccag cgcccacggc agatagggac
     3841 cgaactgtct cacgacgttc taaacccagc tcgcgtacca ctttaaatgg cgaacagcca
     3901 tacccttggg acctacttca gctccaggat gtgatgagcc gacatcgagg tgccaaacac
     3961 cgccgtcgat atgaactctt gggcggtatc agcctgttat ccccggagta ccttttatcc
     4021 gttgagcgat ggcccttcca ttcagaacca ccggatcact atgacctgct ttcgcacctg
     4081 ggatcctcta gagtcgaccg gagaatggcg cgcgccctgt agcggcgcat taagcgcggc
     4141 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     4201 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4261 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4321 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4381 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4441 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4501 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     4561 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4621 atacattcaa atatgtatcc gctcatnnca ngatcctttt taacccatca catatacctg
     4681 ccgttcacta ttatttagtg aaatgagata ttatgatatt ttctgaattg tgattaaaaa
     4741 ggcaacttta tgcccatgca acagaaacta taaaaaatac agagaatgaa aagaaacaga
     4801 tagatttttt agttctttag gcccgtagtc tgcaaatcct tttatgattt tctatcaaac
     4861 aaaagaggaa aatagaccag ttgcaatcca aacgagagtc taatagaatg aggtcgaaaa
     4921 gtaaatcgcg cgggtttgtt actgataaag caggcaagac ctaaaatgtg taaagggcaa
     4981 agtgtatact ttggcgtcac cccttacata ttttaggtct ttttttattg tgcgtaacta
     5041 acttgccatc ttcaaacagg agggctggaa gaagcagacc gctaacacag tacataaaaa
     5101 aggagacatg aacgatgaac atcaaaaagt ttgcaaaaca agcaacagta ttaaccttta
     5161 ctaccgcact gctggcagga ggcgcaactc aagcgtttgc gaaagaaacg aaccaaaagc
     5221 catataagga aacatacggc atttcccata ttacacgcca tgatatgctg caaatccctg
     5281 aacagcaaaa aaatgaaaaa tatcaagttc ctgagttcga ttcgtccaca attaaaaata
     5341 tctcttctgc aaaaggcctg gacgtttggg acagctggcc attacaaaac gctgacggca
     5401 ctgtcgcaaa ctatcacggc taccacatcg tctttgcatt agccggagat cctaaaaatg
     5461 cggatgacac atcgatttac atgttctatc aaaaagtcgg cgaaacttct attgacagct
     5521 ggaaaaacgc tggccgcgtc tttaaagaca gcgacaaatt cgatgcaaat gattctatcc
     5581 taaaagacca aacacaagaa tggtcaggtt cagccacagt tacatctgac ggaaaaatcc
     5641 gtttattcta cactgatttc tccggtaaac attacggcaa acaaacactg acaactgcac
     5701 aagttaacgt atcagcatca gacagctctt tgaacatcaa cggtgtagag gattataaat
     5761 caatctttga cggtgacgga aaaacgtatc aaaatgtaca gcagttcatc gatgaaggca
     5821 actacagctc aggcgacaac catacgctga gagatcctca ctacgtagaa gataaaggcc
     5881 acaaatactt agtatttgaa gcaaacactg gaactgaaga tggctaccaa ggcgaagaat
     5941 ctttatttaa caaagcatac tatggcaaaa gcacatcatt cttccgtcaa gaaagtcaaa
     6001 aacttctgca aagcgataaa aaacgcacgg ctgagttagc aaacggcgct ctcggtatga
     6061 ttgagctaaa cgatgattac acactgaaaa aagtgatgaa accgctgatt gcatctaaca
     6121 cagtaacaga tgaaattgaa cgcgcgaacg tctttaaaat gaacggcaaa tggtacctgt
     6181 tcactgactc ccgcggatca aaaatgacga ttgacggcat tacgtctaac gatatttaca
     6241 tgcttggtta tgtttctaat tctttaactg gcccatacaa gccgctgaac aaaactggcc
     6301 ttgtgttaaa aatggatctt gatcctaacg atgtaacctt tacttactca cacttcgctg
     6361 tacctcaagc gaaaggaaac aatgtcgtga ttacaagcta tatgacaaac agaggattct
     6421 acgcagacaa acaatcaacg tttgcgccaa gcttcctgct gaacatcaaa ggcaagaaaa
     6481 catctgttgt caaagacagc atccttgagc aaggacaatt aacagttaac aaataaaaac
     6541 gcaaaagaaa atgccgatat tgactaccgg aagcagtgtg accgtgtgct tctcaaatgc
     6601 ctgattcagg ctgtctatgt gtgactgttg agctgtaaca agttgtctca ggtgttcaat
     6661 ttcatgttct agttgctttg ttttactggt ttcacctgtt ctgttaggtg ttacatgctg
     6721 ttcatctgtt acattgtcga tctgttcatg gtgaacagct ttaaatgcac caaaaactcg
     6781 taaaagctct gatgtatcta tcttttttac accgttttca tctgtgcata tggacagttt
     6841 tccctttgat atctaacggt gaacagttgt tctacttttg tttgttagtc ttgatgcttc
     6901 actgatagat acaagagcca taagaacctc agatccttcc gtatttagcc agtatgttct
     6961 ctagtgtggt tcgttgtttt tgcgtgagcc atgagaacga accattgaga tcatgcttac
     7021 tttgcatgtc actcaaaaat tttgcctcaa aactggtgag ctgaattttt gcagttaaag
     7081 catcgtgtag tgtttttctt agtccgttac gtaggtagga atctgatgta atggttgttg
     7141 gtattttgtc accattcatt tttatctggt tgttctcaag ttcggttacg agatccattt
     7201 gtctatctag ttcaacttgg aaaatcaacg tatcagtcgg gcggcctcgc ttatcaacca
     7261 ccaatttcat attgctgtaa gtgtttaaat ctttacttat tggtttcaaa acccattggt
     7321 taagcctttt aaactcatgg tagttatttt caagcattaa catgaactta aattcatcaa
     7381 ggctaatctc tatatttgcc ttgtgagttt tcttttgtgt tagttctttt aataaccact
     7441 cataaatcct catagagtat ttgttttcaa aagacttaac atgttccaga ttatatttta
     7501 tgaatttttt taactggaaa agataaggca atatctcttc actaaaaact aattctaatt
     7561 tttcgcttga gaacttggca tagtttgtcc actggaaaat ctcaaagcct ttaaccaaag
     7621 gattcctgat ttccacagtt ctcgtcatca gctctctggt tgctttagct aatacaccat
     7681 aagcattttc cctactgatg ttcatcatct gaacgtattg gttataagtg aacgataccg
     7741 tccgttcttt cctcgtaggg ttttcaatcg tggggttgag tagtgccaca cagcataaaa
     7801 ttagcttggt ttcatgctcc gttaagtcat agcgactaat cgctagttca tttgctttga
     7861 aaacaactaa ttcagacata catctcaatt ggtctaggtg attttaatca ctataccaat
     7921 tgagatgggc tagtcaatga taattactag tccttttcct ttgagttgtg ggtatctgta
     7981 aattctgcta gacctttgct ggaaaacttg taaattctgc tagaccctct gtaaattccg
     8041 ctagaccttt gtgtgttttt tttgtttata ttcaagtggt tataatttat agaataaaga
     8101 aagaataaaa aaagataaaa agaatagatc ccagccctgt gtataactca ctactttagt
     8161 cagttccgca gtattacaaa aggatgtcgt aaacgctgtt tgctcctcta caaaacagac
     8221 cttaaaaccc taaaggctta agtagcaccc tcgcaagctc gggcaaatcg ctgaatattc
     8281 cttctgtctc cgaccatccg gcacctgagt cgctgtcttt ttcgtgacat tcagtccgct
     8341 gcgctcacgg ctctggcagt gaatgggggt aaatggcact acaggcgcct tttatggatt
     8401 catgcaagga aactacccat aatacaagaa aagcccgtca cgggcttctc agggcgtttt
     8461 atggcgggtc tgctatgtgg tgctatctga ctttttgctg ttcagcagtt cctgccctct
     8521 gattttccag tctgaccact tcggattatc ccgtgacagg tcattcagac tggctaatgc
     8581 acccagtaag gcagcggtat catcaacagg cttacccgtc ttactgtcng gatcgacgct
     8641 ctcccttatg cgactcctgc at
//
