LOCUS       Exported                8660 bp ds-DNA     circular SYN 21-JAN-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pKOV
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8660)
  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 8660)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 21, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8660
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      363..585
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             633..1583
                     /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
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     CDS             complement(1976..3397)
                     /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"
     promoter        complement(3398..3843)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     rep_origin      complement(3945..4399)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4082..4103)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4294..4313
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(5009..5026)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5392..5435
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             complement(7631..8290)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGHTTVDISQWHRKEHFEAFQSVARCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     8205..8224
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(8291..8393)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gaactgctga acagcaaaaa gtcagatagc accacatagc agacccgcca taaaacgccc
       61 tgagaagccc gtgacgggct tttcttgtat tatgggtagt ttccttgcat gaatccataa
      121 aaggcgcctg tagtgccatt tacccccatt cactgccaga gccgtgagcg cagcggactg
      181 aatgtcacga aaaagacagc gactcaggtg ccggatggtc ggagacagaa ggaatattca
      241 gcgatttgcc cgagcttgcg agggtgctac ttaagccttt agggttttaa ggtctgtttt
      301 gtagaggagc aaacagcgtt tacgacatcc ttttgtaata ctgcggaact gactaaagta
      361 gtgagttata cacagggctg ggatctattc tttttatctt tttttattct ttctttattc
      421 tataaattat aaccacttga atataaacaa aaaaaacaca caaaggtcta gcggaattta
      481 cagagggtct agcagaattt acaagttttc cagcaaaggt ctagcagaat ttacagatac
      541 ccacaactca aaggaaaagg actagtaatt atcattgact agcccatctc aattggtata
      601 gtgattaaaa tcacctagac caattgagat gtatgtctga attagttgtt ttcaaagcaa
      661 atgaactagc gattagtcgc tatgacttaa cggagcatga aaccaagcta attttatgct
      721 gtgtggcact actcaacccc acgattgaaa accctacgag gaaagaacgg acggtatcgt
      781 tcacttataa ccaatacgtt cagatgatga acatcagtag ggaaaatgct tatggtgtat
      841 tagctaaagc aaccagagag ctgatgacga gaactgtgga aatcaggaat cctttggtta
      901 aaggctttga gattttccag tggacaaact atgccaagtt ctcaagcgaa aaattagaat
      961 tagtttttag tgaagagata ttgccttatc ttttccagtt aaaaaaattc ataaaatata
     1021 atctggaaca tgttaagtct tttgaaaaca aatactctat gaggatttat gagtggttat
     1081 taaaggaact aacacaaaag aaaactcaca aggcaaatat agagattagc cttgatgaat
     1141 ttaagttcat gttaatgctt gaaaataact accatgagtt taaaaggctt aaccaatggg
     1201 ttttgaaacc aataagtaaa gatttaaaca cttacagcaa tatgaaattg gtggttgata
     1261 agcgaggccg cccgactgat acgttgattt tccaagttga actggataga caaatggatc
     1321 tcgtaaccga acttgagaac aaccagataa aaatgaatgg tgacaaaata ccaacaacca
     1381 ttacatcaga ttcctaccta cataacggac taagaaaaac actacacgat gctttaactg
     1441 caaaaattca gctcaccagt tttgaggcaa aatttttgag tgacatgcaa agtaagtatg
     1501 atctcaatgg ttcgttctca tggctcacgc aaaaacaacg aaccacacta gagaacatac
     1561 tggctaaata cggaaggatc tgaggttctt atggctcttg tatctatcag tgaagcatca
     1621 agactaacaa acaaaagtag aacaactgtt caccgttaca tatcaaaggg aaaactgtcc
     1681 atatgcacag atgaaaacgg tgtaaaaaag atagatacat cagagctttt acgagttttt
     1741 ggtgcattta aagctgttca ccatgaacag atcgacaatg taacagatga acagcatgta
     1801 acacctaaca gaacaggtga aaccagtaaa acaaagcaac tagaacatga aattgaacac
     1861 ctgagacaac ttgttacagc tcaacagtca cacatagaca gcctgaatca ggcatttgag
     1921 aagcacacgg tcacactgct tccggtagtc aatatcggca ttttcttttg cgtttttatt
     1981 tgttaactgt taattgtcct tgctcaagga tgctgtcttt gacaacagat gttttcttgc
     2041 ctttgatgtt cagcaggaag cttggcgcaa acgttgattg tttgtctgcg tagaatcctc
     2101 tgtttgtcat atagcttgta atcacgacat tgtttccttt cgcttgaggt acagcgaagt
     2161 gtgagtaagt aaaggttaca tcgttaggat caagatccat ttttaacaca aggccagttt
     2221 tgttcagcgg cttgtatggg ccagttaaag aattagaaac ataaccaagc atgtaaatat
     2281 cgttagacgt aatgccgtca atcgtcattt ttgatccgcg ggagtcagtg aacaggtacc
     2341 atttgccgtt cattttaaag acgttcgcgc gttcaatttc atctgttact gtgttagatg
     2401 caatcagcgg tttcatcact tttttcagtg tgtaatcatc gtttagctca atcataccga
     2461 gagcgccgtt tgctaactca gccgtgcgtt ttttatcgct ttgcagaagt ttttgacttt
     2521 cttgacggaa gaatgatgtg cttttgccat agtatgcttt gttaaataaa gattcttcgc
     2581 cttggtagcc atcttcagtt ccagtgtttg cttcaaatac taagtatttg tggcctttat
     2641 cttctacgta gtgaggatct ctcagcgtat ggttgtcgcc tgagctgtag ttgccttcat
     2701 cgatgaactg ctgtacattt tgatacgttt ttccgtcacc gtcaaagatt gatttataat
     2761 cctctacacc gttgatgttc aaagagctgt ctgatgctga tacgttaact tgtgcagttg
     2821 tcagtgtttg tttgccgtaa tgtttaccgg agaaatcagt gtagaataaa cggatttttc
     2881 cgtcagatgt aactgtggct gaacctgacc attcttgtgt ttggtctttt aggatagaat
     2941 catttgcatc gaatttgtcg ctgtctttaa agacgcggcc agcgtttttc cagctgtcaa
     3001 tagaagtttc gccgactttt tgatagaaca tgtaaatcga tgtgtcatcc gcatttttag
     3061 gatctccggc taatgcaaag acgatgtggt agccgtgata gtttgcgaca gtgccgtcag
     3121 cgttttgtaa tggccagctg tcccaaacgt ccaggccttt tgcagaagag atatttttaa
     3181 ttgtggacga atcgaactca ggaacttgat atttttcatt tttttgctgt tcagggattt
     3241 gcagcatatc atggcgtgta atatgggaaa tgccgtatgt ttccttatat ggcttttggt
     3301 tcgtttcttt cgcaaacgct tgagttgcgc ctcctgccag cagtgcggta gtaaaggtta
     3361 atactgttgc ttgttttgca aactttttga tgttcatcgt tcatgtctcc ttttttatgt
     3421 actgtgttag cggtctgctt cttccagccc tcctgtttga agatggcaag ttagttacgc
     3481 acaataaaaa aagacctaaa atatgtaagg ggtgacgcca aagtatacac tttgcccttt
     3541 acacatttta ggtcttgcct gctttatcag taacaaaccc gcgcgattta cttttcgacc
     3601 tcattctatt agactctcgt ttggattgca actggtctat tttcctcttt tgtttgatag
     3661 aaaatcataa aaggatttgc agactacggg cctaaagaac taaaaaatct atctgtttct
     3721 tttcattctc tgtatttttt atagtttctg ttgcatgggc ataaagttgc ctttttaatc
     3781 acaattcaga aaatatcata atatctcatt tcactaaata atagtgaacg gcaggtatat
     3841 gtgatgggtt aaaaaggatc gcggtgagcg gatacatatt tgaatgtatt tagaaaaata
     3901 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa
     3961 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     4021 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     4081 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     4141 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     4201 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     4261 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     4321 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     4381 tgcgccgcta cagggcgcgc gccattctcc ggtcgactct agaggatccc aggtgcgaaa
     4441 gcaggtcata gtgatccggt ggttctgaat ggaagggcca tcgctcaacg gataaaaggt
     4501 actccgggga taacaggctg ataccgccca agagttcata tcgacggcgg tgtttggcac
     4561 ctcgatgtcg gctcatcaca tcctggagct gaagtaggtc ccaagggtat ggctgttcgc
     4621 catttaaagt ggtacgcgag ctgggtttag aacgtcgtga gacagttcgg tccctatctg
     4681 ccgtgggcgc tggagaactg aggggggctg ctcctagtac gagaggaccg gagtggacgc
     4741 atcactggtg ttcgggttgt catgccaatg gcactgcccg gtagctaaat gcggaagaga
     4801 taagtgctga aagcatctaa gcacgaaact tgccccgaga tgagttctcc ctgactcctt
     4861 gagagtcctg aaggaacgtt gaagacgacg acgttgatag gccgggtgtg taagcgcagc
     4921 gatgcgttga gctaaccggt actaatgaac cgtgaggctt aaccttacaa cgccgaagat
     4981 gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg
     5041 gtctgatgaa acagaatttg cctggcggcc gtagcgcggt ggtcccacct gaccccatgc
     5101 cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag
     5161 tagggaactg ccaggcatca aattaagcag taagccggtc ataaaaccgg tggttgtaaa
     5221 agaattcggt ggagcggtag ttcagtcggt tagaatacct gcctgtcacg cagggggtcg
     5281 cgggttcgag tcccgtccgt tccgccaccc taattagggg cgtagttcaa ttggtagggc
     5341 accggtctcc aaaaccgggt gttgggagtt cgagtctctc cgcccctgcc agaaatcatc
     5401 cttagcgaaa gctaaggatt ttttttatct gaaataaccc tctccgaagt aaatccttct
     5461 accggcatcc ttgccagcca ttcatattaa tacacttcat ccagcacgtt aattttcaaa
     5521 agatcgcgaa tcaacgcatt tttatcgcta ttttgcagcc atatggcata aagcggccgt
     5581 gaaagtgttg tgctatcgac aacggtatgc aggccgcctt ttttacgcgc ccagctgacg
     5641 ggtagccagg tgcaaccatt aagcatcgca atctgttgct gtgccagttc agcagaactg
     5701 gttgtcagaa tgggcacttc gtcagcaccg atcaaacctg cctcatgctg ttgaaaatct
     5761 ggcccccact caagtcgcag ataattaaga tctcccttta gttttgaagg ggcactggta
     5821 taaagcgcta aagtgaaata tcccagcaac tgactactaa attcgtccat tttgggcgct
     5881 tcagtggtaa taagaagatc aagctggcgt tcatgcagct gttttaccac acgcaataac
     5941 cttcacactc caaatttata acatccacag tgctctccgc aaaattattt gtcgttatga
     6001 tttaaatgtt ttgttttaca ctctgtcaag cgtaactaat actccgcgcc ataactagct
     6061 cggtcaaaga attaggagcg tgcaggatgg cggaaagctt tacgacgact aatcgatatt
     6121 tcgacaataa acattatcca cgtggattct ctcgtcatgg tgatttcacc atcaaagagg
     6181 cacaactgct tgagcgtcat ggttatgcct tcaatgagtt ggatcttggc aaacgcgagc
     6241 cggttaccga ggaagagaaa ctcttcgtag cagtatgccg tggcgaacgt gagccagtga
     6301 cagaagcaga acgcgtgtgg tccaagtata tgacgcgtat taagcgtcca aaacgttttc
     6361 acaccctttc cggcggtaaa ccgcaggttg aaggtgctga agactacacc gattctgacg
     6421 attaataaaa agggcgaaat gccctttttt tatgtcagta gtttctgcag atggatgagc
     6481 aaacggtcaa tcgctcgata gctaactgcc tcctgcaaat gctgacgtgt gataatgtca
     6541 gactgatcaa tatcagcaat ggttcgtgca actttcaata accgctgcca ggcacgaatc
     6601 gataacccca gatggatcag tgttccttcc aaccacatcg catcttcgct ctcaagtttg
     6661 cagaattggc gtatttccgg actatccagc caggcattca gtttattctg ccgcttaaat
     6721 tggcgctctc tggcggccat tacgcgttgt ttaatggtgg cgctgctttc tcccggcact
     6781 accgttttac tcaaagtgcc ggggggtggt aatgggatct ccagtgagag atcgaagcgg
     6841 tcgagaaagg gccccgagag ccggttgaga taacgtaatg tctgttctgg cgtgcagcgg
     6901 ttatggtttc cctgataatg cccggtaggg ctgggattca tcgccgcaac aagctggaaa
     6961 cgggctggat aggttatttt tgctcgtgcg cgtgaaagat ggatctgccc ggattcaatc
     7021 ggctctcgca aggcatccgg tgtacgccgt tcaaattcag gtagctcatc aagaaaaagc
     7081 acgccgttat gcgccagcga aatttcaccg ggccctggaa ttgcgccacc gcctaccatc
     7141 gcagttaacg atgcactgtg atgaggtgag cggaacgggc gctgccgcca ttgtttttgt
     7201 actgattcag catttaccag acttaatatc gcagcactct ccagtgcctc ttcattgctt
     7261 aaatctggca acaggccatt aatacggctg gcgagcattg ttttacctgt tcccggcggc
     7321 ccaatcagta aaaggttgtg cccgccagca gcggtaattt ccagtcctcg ctttccttgt
     7381 tcctgaccga taacatcact gagatcatgt tgtagcgccc gggatactgc atcagttggg
     7441 ttcggggcgg ccgcgatccc cgggtaccga ttaaaccagc aatagacata agcggctatt
     7501 taacgaccct gccctgaacc gacgaccggg tcgaatttgc tttcgaattt ctgccattca
     7561 tccgcttatt atcacttatt caggcgtagc aaccaggcgt ttaagggcac caataactgc
     7621 cttaaaaaaa ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca
     7681 ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca
     7741 gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt
     7801 ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga
     7861 aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca
     7921 catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg
     7981 atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata
     8041 tcaccagctc accgtctttc attgccatac ggaattccgg atgagcattc atcaggcggg
     8101 caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa
     8161 aggccgtaat atccagctga acggtctggt tataggtaca tcgagcaact gactgaaatg
     8221 cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatgt ccagtgattt
     8281 ttttctccat tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg
     8341 gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc
     8401 attttcgcca aaagttggcc cagggcttcc cggtatcaac agggacacca ggatttattt
     8461 actctgcgaa gtgatcttcc gtcacaggta tttattcgaa gacgaaaggt gcaggagtcg
     8521 cataagggag agcgtcgatc cccgacagta agacgggtaa gcctgttgat gataccgctg
     8581 ccttactggg tgcattagcc agtctgaatg acctgtcacg ggataatccg aagtggtcag
     8641 actggaaaat cagagggcag
//
