LOCUS       Exported                6671 bp DNA     circular SYN 08-OCT-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6671)
  AUTHORS   Kyle Mohler
  TITLE     Direct Submission
  JOURNAL   Exported Oct 12, 2022 from SnapGene 6.0.6
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6671
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     primer_bind     228..279
                     /label=KM167
     rep_origin      complement(291..879)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(896..944)
                     /label=KM168
     CDS             complement(920..1257)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPS"
     primer_bind     1462..1478
                     /label=KM142
     primer_bind     1497..1559
                     /label=glnS_E45MCS_F
     promoter        1523..1608
                     /label=glnS*
                     /note="Mutant E. coli glnS promoter from Schultz lab"
     primer_bind     complement(1543..1607)
                     /label=glnSR
     primer_bind     1578..1637
                     /label=SepRS_glnS_F1 (KM15)
     CDS             1617..3235
                     /codon_start=1
                     /label=SepRS
                     /translation="AMFKREEIIEMANKDFEKAWIETKDLIKAKKINESYPRIKPVFGK
                     THPVNDTIENLRQAYLRMGFEEYINPVIVDERDIYKQFGPEAMAVLDRCFYLAGLPRPD
                     VGLSDEKISQIEKLGIKVSEHKESLQKILHGYKKGTLDGDDLVLEISNALEISSEMGLK
                     ILEDVFPEFKDLTAVSSKLTLRSHMTSGWFLTVSDLMNKKPLPFKLFSIDRCFRREQKE
                     DKSHLMTYHSASCAIAGEGVDINDGKAIAEGLLSQFGFTNFKFIPDEKKSKYYTPETQT
                     EVYAYHPKLKEWLEVATFGVYSPVALSKYGIDVPVMNLGLGVERLAMISGNFADVREMV
                     YPQFYEHELNDRDVASMVKLDKVPVMDEIYDLTKELIESCVKNKDLKSPCELAIEKTFS
                     FGKTKKNVKINIFSKIEGKNLLGPSILNEIYVYDGNVIGIPESFDGVKEEFKDFLEKGK
                     SEGVATGIRYIDALCFKITSKLEEAFVSNTTEFKVKVRRVRSLSDINLKIDDIAIKQIM
                     SKNKVIDVRGPVFLNVEVKIE"
     primer_bind     2291..2315
                     /label=SepRS Repair_F (KM130)
     RBS             3272..3277
     CDS             3295..4479
                     /codon_start=1
                     /label=EF-Sep
                     /translation="MSKEKFERTKPHVNVGTIGHVDHGKTTLTAAITTVLAKTYGGAAR
                     AFDQIDNAPEEKARGITINTSRVEYDTPTRHYAHVDCPGHADYVKNMITGAAQMDGAIL
                     VVAATDGPMPQTREHILLGRQVGVPYIIVFLNKCDMVDDEELLELVEMEVRELLSQYDF
                     PGDDTPIVRGSALKALEGDAEWEAKILELAGFLDSYIPEPERAIDKPFLLPIVGVYSIS
                     GRGTVVSGRVERGIIKVGEEVEIVGIKETQKSTCTGVEMFRKLLDEGRAGEWVGVLLRG
                     IKREEIERGQVLAKPGTIKPHTKFESEVYILSKDEGGRHTPFFKGYRPQFYFRTTDVTG
                     TIELPEGVEMVMPGDNIKMVVTLIHPIAMDDGLRFAIREGGRTVGAGVVAKVLS"
     primer_bind     complement(3340..3360)
                     /label=SepRS Repair_R (KM131)
     primer_bind     4458..4494
                     /label=KM210
     terminator      4715..4801
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4893..4920
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     4914..4937
                     /label=FRT-KAN_N45_F (KM140)
     primer_bind     4929..4979
                     /label=Fragment 1.FOR
     promoter        4953..5014
                     /label=proK promoter
     tRNA            5015..5089
                     /label=Sep tRNA A36 (G2:C71)
     terminator      5090..5124
                     /label=proK term
     primer_bind     complement(5104..5134)
                     /label=Fragment 1.REV
     primer_bind     5115..5150
                     /label=Fragment 2.FOR
     terminator      5124..5132
                     /label=rrnB T2
     primer_bind     5126..5171
                     /label=FRT-KAN_N45_F (KM140)
     protein_bind    complement(5176..5223)
                     /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)."
     CDS             5584..6378
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     protein_bind    complement(6569..6602)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(6617..6667)
                     /label=FRT-KAN_N45_R (KM141)
     primer_bind     complement(6617..6642)
                     /label=KM143
     primer_bind     complement(6648..6670)
                     /label=Fragment 2.REV
     primer_bind     complement(join(6649..6671,1..20))
                     /label=Fragment 2.REV
     primer_bind     complement(join(6669..6671,1..17))
                     /label=FRT-KAN_N45_R (KM141)
ORIGIN
        1 ctagcggagt gtatactggc ttactatgtt ggcactgatg agggtgtcag tgaagtgctt
       61 catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga tacaggatat
      121 attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa
      181 tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt aacagggaag
      241 tgagagggcc gcggggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
      301 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
      361 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
      421 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
      481 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
      541 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
      601 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
      661 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
      721 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
      781 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
      841 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
      901 acggggtctg acgctcaata ttgacggcca gttaagccat tcatgccagt aggcgcgcgg
      961 acgaaagtaa acccactggt gataccattc gcgagcctcc ggatgacgac cgtagtgatg
     1021 aatctctcct ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt ctcgtccctg
     1081 atttttcacc accccctgac cgcgaatggt gagattgaga atataacctt tcattcccag
     1141 cggtcggtcg ataaaaaaat cgagataacc gttggcctca atcggcgtta aacccgccac
     1201 cagatgggca ttaaacgagt atcccggcag caggggatca ttttgcgctt cagccatact
     1261 tttcatactc ccgccattca gagaagaaac caattgtcca tattgcatca gacattgccg
     1321 tcactgcgtc ttttactggc tcttctcgct aaccaaaccg gtaaccccgc ttattaaaag
     1381 cattctgtaa caaagcggga ccaaagccat gacaaaaacg cgtaacaaaa gtgtctataa
     1441 tcacggcaga aaagtccaca ttgattattt gcacggcgtc acactttgct atgccatagc
     1501 atttttatcc ataagattag cgcgattatc aattttaaaa aactaacagt tgtcagcctg
     1561 tcccgcttta atatcatacg ccgttatacg ttgtttacgc tttgaggaat ccacgatggc
     1621 aatgtttaaa agagaagaaa tcattgaaat ggccaataag gactttgaaa aagcatggat
     1681 cgaaactaaa gaccttataa aagctaaaaa gataaacgaa agttacccaa gaataaaacc
     1741 agtttttgga aaaacacacc ctgtaaatga cactattgaa aatttaagac aggcatatct
     1801 tagaatgggt tttgaagaat atataaaccc agtaattgtc gatgaaagag atatttataa
     1861 acaattcggc ccagaagcta tggcagtttt ggatagatgc ttttatttag cgggacttcc
     1921 aagacctgac gttggtttga gcgatgaaaa aatttcacag attgaaaaac ttggaattaa
     1981 agtttctgag cacaaagaaa gtttacaaaa aatacttcac ggatacaaaa aaggaactct
     2041 tgatggtgac gatttagttt tagaaatttc aaatgcactt gaaatttcaa gcgagatggg
     2101 tttaaaaatt ttagaagatg ttttcccaga atttaaggat ttaaccgcag tttcttcaaa
     2161 attaacttta agaagccaca tgacttcagg atggttcctt actgtttcag acctcatgaa
     2221 caaaaaaccc ttgccattta aactcttttc aatcgataga tgttttagaa gagaacaaaa
     2281 agaagataaa agccacttaa tgacatacca ctctgcatcc tgtgcaattg caggtgaagg
     2341 cgtggatatt aatgatggaa aagcaattgc agaaggatta ttatcccaat ttggctttac
     2401 aaactttaaa ttcattcctg atgaaaagaa aagtaaatac tacacccctg aaacacagac
     2461 tgaagtttac gcataccacc caaaattaaa agaatggctc gaagttgcta catttggagt
     2521 atattcgcca gttgcattaa gcaaatacgg aatagatgta cctgtaatga atttgggtct
     2581 tggtgttgaa agacttgcaa tgatttctgg aaatttcgca gatgttcgag aaatggtata
     2641 tcctcagttt tacgaacacg aacttaatga ccgggatgtc gcttcaatgg taaaactcga
     2701 taaagttcca gtaatggatg aaatttacga tttaacaaaa gaattaattg agtcatgtgt
     2761 taaaaacaaa gatttaaaat ccccttgtga attagctatt gaaaaaacgt tttcatttgg
     2821 aaaaaccaag aaaaatgtaa aaataaacat tttttcaaaa atagaaggta aaaatttact
     2881 cggaccttca attttaaacg aaatctacgt ttacgatgga aatgtaattg gaattcctga
     2941 aagctttgac ggagtaaaag aagaatttaa agacttctta gaaaaaggaa aatcagaagg
     3001 ggtagcaaca ggcattcgat atatcgatgc gctttgcttt aaaattactt caaaattaga
     3061 agaagcattt gtgtcaaaca ctactgaatt caaagttaaa gttcgacgtg tcagaagttt
     3121 aagcgacatt aacttaaaaa tcgatgatat cgcaataaaa cagatcatga gcaaaaataa
     3181 agtaatcgac gttagaggcc cagtcttttt aaatgtcgaa gtaaaaattg aataagagct
     3241 cggtacctaa agtatattag ttaagtataa gaaggagata tacatatggg atccatgtct
     3301 aaagaaaagt ttgaacgtac aaaaccgcac gttaacgtcg gtactatcgg ccacgttgac
     3361 catggtaaaa caacgctgac cgctgcaatc actaccgtac tggctaaaac ctacggcggt
     3421 gctgctcgcg cattcgacca gatcgataac gcgccggaag aaaaagctcg tggtatcacc
     3481 atcaacactt ctcgggttga atacgacacc ccgacccgtc actacgcaca cgtagactgc
     3541 ccggggcacg ccgactatgt taaaaacatg atcaccggtg ctgcgcagat ggacggcgcg
     3601 atcctggtag ttgctgcgac tgacggcccg atgccgcaga ctcgtgagca catcctgctg
     3661 ggtcgtcagg taggcgttcc gtacatcatc gtgttcctga acaaatgcga catggttgat
     3721 gacgaagagc tgctggaact ggttgaaatg gaagttcgtg aacttctgtc tcagtacgac
     3781 ttcccgggcg acgacactcc gatcgttcgt ggttctgctc tgaaagcgct ggaaggcgac
     3841 gcagagtggg aagcgaaaat cctggaactg gctggcttcc tggattctta cattccggaa
     3901 ccagagcgtg cgattgacaa gccgttcctg ctgccgatcg tcggggtata ctccatctcc
     3961 ggtcgtggta ccgttgtttc gggtcgtgta gaacgcggta tcatcaaagt tggtgaagaa
     4021 gttgaaatcg ttggtatcaa agagactcag aagtctacct gtactggcgt tgaaatgttc
     4081 cgcaaactgc tggacgaagg ccgtgctggt gagtgggtag gtgttctgct gcgtggtatc
     4141 aaacgtgaag aaatcgaacg tggtcaggta ctggctaagc cgggcaccat caagccgcac
     4201 accaagttcg aatctgaagt gtacattctg tccaaagatg aaggcggccg tcatactccg
     4261 ttcttcaaag gctaccgtcc gcagttctac ttccgtacta ctgacgtgac tggtaccatc
     4321 gaactgccgg aaggcgtaga gatggtaatg ccgggcgaca acatcaaaat ggttgttacc
     4381 ctgatccacc cgatcgcgat ggacgacggt ctgcgtttcg caatccgtga aggcggccgt
     4441 accgttggcg cgggcgttgt agcaaaagtt ctgagctaat ggcgcgccgt cgacctgcag
     4501 gcatgcaagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
     4561 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
     4621 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
     4681 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     4741 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     4801 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     4861 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt
     4921 tgcgtttcta caaactcttt ttgtttgcta gcaggcattt tgctattaag ggattgacga
     4981 gggcgtatct gcgcagtaag atgcgccccg cattggcggg gtagtctagg ggttaggcag
     5041 cggactctaa atccgcctta cgtgggttca aatcccaccc ccgcctccaa attcgaaaag
     5101 cctgctcaac gagcaggctt ttttggcctt tttgcgtttc tacaaactcc cgattgtgta
     5161 ggctggagct gcttcgaagt tcctatactt tctagagaat aggaacttcg gaataggaac
     5221 ttcaagatcc cctcacgctg ccgcaagcac tcagggcgca agggctgcta aaggaagcgg
     5281 aacacgtaga aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg
     5341 gctatctgga caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt
     5401 acatggcgat agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct
     5461 ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg
     5521 ccaaggatct gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt
     5581 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     5641 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     5701 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     5761 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     5821 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     5881 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     5941 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     6001 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     6061 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     6121 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     6181 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     6241 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     6301 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     6361 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     6421 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     6481 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     6541 tcgcccaccc cagcttcaaa agcgctctga agttcctata ctttctagag aataggaact
     6601 tcggaatagg aactaaggag gatattcata tggaccatgg ctgatcagcg ctagcggagt
     6661 gtatactggc g
//
