LOCUS       Exported                7373 bp DNA     circular SYN 26-JUL-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7373)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 26, 2021 from SnapGene Viewer 5.3.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7373
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    6..130
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        155..185
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             239..919
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     CDS             1239..1544
                     /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"
     protein_bind    complement(1585..1709)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1744..1760)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(1790..1808)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1829..1845)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1853..1869
                     /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(1877..1907)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1922..1943
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2231..2819)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2936..3736)
                     /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            4052..4161
                     /note="incP origin of transfer"
     regulatory      4736..4745
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4742..6025
                     /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"
     CDS             6047..6676
                     /codon_start=1
                     /label=upp
                     /translation="MAKIVEVKHPLVKHKLGLMREQDISTKRFRELASEVGSLLTYEAT
                     ADLETEKVTIEGWNGPVEIDQIKGKKITVVPILRAGLGMMDGVLENVPSARISVVGMYR
                     NEETLEPVPYFQKLVSNIDERMALIVDPMLATGGSVIATIDLLKKAGCSSIKVLVLVAA
                     PEGIAALEKAHPDVELYTASIDQGLNEHGYIIPGLGDAGDKIFGTK"
     rep_origin      complement(6706..7134)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     7276..7292
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        7302..7320
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     7353..7369
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 atcaaacaag tttgtacaaa aaagctgaac gagaaacgta aaatgatata aatatcaata
       61 tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca caacatatcc
      121 agtcactatg gcggccgcat taggcacccc aggctttaca ctttatgctt ccggctcgta
      181 taatgtgtgg attttgagtt aggatccgtc gagattttca ggagctaagg aagctaaaat
      241 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca
      301 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat
      361 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
      421 cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga aagacggtga
      481 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac
      541 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc
      601 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa
      661 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
      721 caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga
      781 caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt
      841 cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcaggcgg ggcgtaatct
      901 agaggatccg gcttactaaa agccagataa cagtatgcgt atttgcgcgc tgatttttgc
      961 ggtataagaa tatatactga tatgtatacc cgaagtatgt caaaaagagg tatgctatga
     1021 agcagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga
     1081 tgtcaatatc tccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc
     1141 cgaacgctgg aaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat
     1201 gaacggctct tttgctgacg agaacagggg ctggtgaaat gcagtttaag gtttacacct
     1261 ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc
     1321 ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtccccc
     1381 gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata
     1441 tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa
     1501 atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca ggctccctta
     1561 tacacagcca gtctgcaggt cgaccatagt gactggatat gttgtgtttt acagtattat
     1621 gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt
     1681 tctcgttcag ctttcttgta caaagtggtt cgatgggctg caggaattcg atatcaagct
     1741 tatcgatacc gtcgacctcg agggggggcc cggtacccag cttttgttcc ctttagtgag
     1801 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     1861 cgctcacaat tccacacaac atacgagccg ggagcataaa gtgtaaagcc tggggtgcct
     1921 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     1981 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2041 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2101 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2161 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2221 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2281 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2341 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2401 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     2461 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     2521 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2581 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     2641 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     2701 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     2761 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     2821 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     2881 ggattttggt catgactaga gaataggaac ttcggaatag gaacttatga gctcagccaa
     2941 tcgactggcg agcggcatcg cattcttcgc atcccgcctc tggcggatgc aggaagatca
     3001 acggatctcg gcccagttga cccagggctg tcgccacaat gtcgcgggag cggatcaacc
     3061 gagcaaaggc atgaccgact ggaccttcct tctgaaggct cttctccttg agccacctgt
     3121 ccgccaaggc aaagcgctca cagcagtggt cattctcgag ataatcgacg cgtaccaact
     3181 tgccatcctg aagaatggtg cagtgtctcg gcaccccata gggaaccttt gccatcaact
     3241 cggcaagatg cagcgtcgtg ttggcatcgt gtcccacgcc gaggagaagt acctgcccat
     3301 cgagttcatg gacacgggcg accgggcttg caggcgagtg aggtggcagg ggcaatggat
     3361 cagagatgat ctgctctgcc tgtggccccg ctgccgcaaa ggcaaatgga tgggcgctgc
     3421 gctttacatt tggcaggcgc cagaatgtgt cagagacaac tccaaggtcc ggtgtaacgg
     3481 gcgacgtggc aggatcgaac ggctcgtcgt ccagacctga ccacgagggc atgacgagcg
     3541 tccctcccgg acccagcgca gcacgcaggg cctcgatcag tccaagtggc ccatcttcga
     3601 ggggccggac gctacggaag gagctgtgga ccagcagcac accgccgggg gtaaccccaa
     3661 ggttgagaag ctgaccgatg agctcggctt ttcgccattc gtattgcacg acattgcact
     3721 ccaccgctga tgacatcagt cgatcatagc acgatcaacg gcactgttgc aaatagtcgg
     3781 tggtgataaa cttatcatcc ccttttgctt atggagctgc acatgaaccc attcaaaggc
     3841 cggcattttc agcgtgacat cattctgtgg gccgtacgct ggtactgcaa atacggcatc
     3901 agttaccgtg agctgcattt tccgctgcat aaccctgctt cggggtcatt atagcgattt
     3961 tttcggtata tccatccttt ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag
     4021 actttccttg gtgtatccaa cggcgtcagc cgggcaggat aggtgaagta ggcccacccg
     4081 cgagcgggtg ttccttcttc actgtccctt attcgcacct ggcggtgctc aacgggaatc
     4141 ctgctctgcg aggctggcgg gaacttcgaa gttcctatac tttctaggca tgagcggata
     4201 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     4261 agtgccacct aaattgtaag cgttaataat tcgatagctt cacgctgccg caagcactca
     4321 gggcgcaagg gctgctaaag gaagcggaac acgtagaaag ccagtccgca gaaacggtgc
     4381 tgaccccgga tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag
     4441 agaaagcagg tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg
     4501 acagcaagcg aaccggaatt gccagctggg gcgccctctg gtaaggtttg ggaagccctg
     4561 caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc
     4621 gatctgatca agagacagga tgaggatcgt ttcgcatgat atcatcaagc ttatcgatac
     4681 cgtcgactac taaccttctt ctctttccta cagctgagat caccggcgaa ggagggccac
     4741 catggtgtcg aataacgctt tagaaacaat tattaacgcc cggttaccag gcgaagaggg
     4801 gctgtggcag attcatctgc aggacggaaa aatcagcgcc attgatgcgc aatccggcgt
     4861 gatgcccata actgaaaaca gcctggatgc cgaacaaggt ttagttatac cgccgtttgt
     4921 ggagccacat attcacctgg acaccacgca aaccgccgga caaccgaact ggaatcagtc
     4981 cggcacgctg tttgaaggca ttgaacgctg ggccgagcgc aaagcgttat taacccatga
     5041 cgatgtgaaa caacgcgcat ggcaaacgct gaaatggcag attgccaacg gcattcagca
     5101 tgtgcgtacc catgtcgatg tttcggatgc aacgctaact gcgctgaaag caatgctgga
     5161 agtgaagcag gaagtcgcgc cgtggattga tctgcaaatc gtcgccttcc ctcaggaagg
     5221 gattttgtcg tatcccaacg gtgaagcgtt gctggaagag gcgttacgct taggggcaga
     5281 tgtagtgggg gcgattccgc attttgaatt tacccgtgaa tacggcgtgg agtcgctgca
     5341 taaaaccttc gccctggcgc aaaaatacga ccgtctcatc gacgttcact gtgatgagat
     5401 cgatgacgag cagtcgcgct ttgtcgaaac cgttgctgcc ctggcgcacc atgaaggcat
     5461 gggcgcgcga gtcaccgcca gccacaccac ggcaatgcac tcctataacg gggcgtatac
     5521 ctcacgcctg ttccgcttgc tgaaaatgtc cggtattaac tttgtcgcca acccgctggt
     5581 caatattcat ctgcaaggac gtttcgatac gtatccaaaa cgtcgcggca tcacgcgcgt
     5641 taaagagatg ctggagtccg gcattaacgt ctgctttggt cacgatgatg tcttcgatcc
     5701 gtggtatccg ctgggaacgg cgaatatgct gcaagtgctg catatggggc tgcatgtttg
     5761 ccagttgatg ggctacgggc agattaacga tggcctgaat ttaatcaccc accacagcgc
     5821 aaggacgttg aatttgcagg attacggcat tgccgccgga aacagcgcca acctgattat
     5881 cctgccggct gaaaatgggt ttgatgcgct gcgccgtcag gttccggtac gttattcggt
     5941 acgtggcggc aaggtgattg ccagcacaca accggcacaa accaccgtat atctggagca
     6001 gccagaagcc atcgattaca aacgtatcag cggagctaat ggcgtcatgg ctaagatcgt
     6061 ggaagtcaaa cacccactcg tcaaacacaa gctgggactg atgcgtgagc aagatatcag
     6121 caccaagcgc tttcgcgaac tcgcttccga agtgggtagc ctgctgactt acgaagcgac
     6181 cgccgacctc gaaacggaaa aagtaactat cgaaggctgg aacggcccgg tagaaatcga
     6241 ccagatcaaa ggtaagaaaa ttaccgttgt gccaattctg cgtgcgggtc ttggtatgat
     6301 ggacggtgtg ctggaaaacg ttccgagcgc gcgcatcagc gttgtcggta tgtaccgtaa
     6361 tgaagaaacg ctggagccgg taccgtactt ccagaaactg gtttctaaca tcgatgagcg
     6421 tatggcgctg atcgttgacc caatgctggc aaccggtggt tccgttatcg cgaccatcga
     6481 cctgctgaaa aaagcgggct gcagcagcat caaagttctg gtgctggtag ctgcgccaga
     6541 aggtatcgct gcgctggaaa aagcgcaccc ggacgtcgaa ctgtataccg catcgattga
     6601 tcagggactg aacgagcacg gatacattat tccgggcctc ggcgatgccg gtgacaaaat
     6661 ctttggtacg aaataagctt gtcgaaattc gctagatttt gttaaaattc gcgttaaatt
     6721 cttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     6781 caaaagaata gaccgagata gggttgagtg ttgttccagt ctggaacaag agtccactat
     6841 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     6901 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     6961 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     7021 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     7081 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     7141 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     7201 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     7261 cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg
     7321 gcgaattgga gctccaccgc ggtggcggcc gctctagaac tagtggatcc ccc
//
