LOCUS       Exported                7197 bp ds-DNA     circular SYN 03-APR-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7197)
  AUTHORS   Anne Ruffing
  TITLE     Direct Submission
  JOURNAL   Exported Nov 13, 2020 from SnapGene 4.1.9
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7197
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    397..1271
                     /label=NS1_Davies_5
                     /note="homology region for insertion at NS1 from Davies 
                     2014 publication"
     terminator      1284..1311
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1452..2069)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
     primer_bind     1977..1996
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     RBS             2076..2087
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    2175..2192
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(2176..2205)
                     /label=pCas34i_R
     misc_RNA        2232..2307
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     2232..2259
                     /label=pCas34i_F
     CDS             2328..2357
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2373..2390
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2446..2463)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     primer_bind     2546..2579
                     /label=SpRCas_F
     primer_bind     complement(2546..2563)
                     /label=pCas34CmR
     primer_bind     complement(2790..2807)
                     /label=PSpRmR
     primer_bind     2808..2834
                     /label=PSpRmF
     CDS             3282..4073
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SpR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     primer_bind     complement(4428..4475)
                     /label=SpRCas_R
     misc_feature    4453..4957
                     /label=NS1-Davies-3
                     /note="homology region for insertion at NS1 from Davies 
                     2014 paper"
     primer_bind     4453..4477
                     /label=pCas34Cm_F
     primer_bind     complement(4976..4992)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4976..4992)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4989..5011)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5000..5016
                     /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(5024..5054)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5069..5090
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5207..5224)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5378..5966)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5458..5477)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6137..6997)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6760..6779
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6998..7102)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7170..7188
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgatgt ttggtcgaaa attcttcaca
      421 cttttaacgg ctgtctccac agtgttcctg actcctattt acggagcggc tcaaacgatg
      481 caagatcaaa gtaatacgat gaataatgaa aattcattgg ggaatagtgc gactggggta
      541 gtcaaccaaa ccgctgtgta caacttaggt caaagtgcca cctacggttt tagagggatc
      601 gtttgtcccc gcccttcctt tgtgctcagt ggctctgcaa atggcaatac atttgatggg
      661 gagagttcaa attcctacgc tctttccggt gccattattg tgcccctcgg tggtcaagtg
      721 ggcagcaact gtagagattt cagcactacc ctactggaaa gagaacaagc cgaattagca
      781 aagtttcaaa tccaatcttc agcggaaaca gtcaaacttt gtgctgacat gatttcttca
      841 ggcattcaag tagatccaga ggtttatcca gtactggcgc ggcaatgttc tgcagttcgg
      901 ttggtttcac aaggacgggc ttctagcaca aatgaagccc gacaaaatga atcgactcac
      961 aataagctag atgatgatgt agaattgtct gctaattact taaaaactga gtaaatataa
     1021 tattttgaat agtgaggaaa aatatctcaa aggctatatg aaaataaaaa aagagcggct
     1081 aaaaaatctt cagcttacag tgacgcctgt ctttgcattc taaagcacta aaacacagtc
     1141 gtttttttac tgactccaag tgtttttatt actatactta gaaattcgtc aaataggcat
     1201 gaaattgctg tattaaaata tcctactgta tcttttagtt cacactattt ttcttgagtt
     1261 aattttgtat caataactat aaacgcagaa aggcccaccc gaaggtgagc cagtgtgact
     1321 ctagtagaga gcgttcaacc ccttggggcc tctaaacggg tcttgagggg ttttttgtct
     1381 agtagtgatc tacactagca ctatcagtgt tattaagcta ctaaagcgta gttttcgtcg
     1441 tttgcagcgg acccactttc acatttaagt tgtttttcta atccgcatat gatcaattca
     1501 aggccgaata agaaggctgg ctctgcacct tggtgatcaa ataattcgat agcttgtcgt
     1561 aataatggcg gcatactatc agtagtaggt gtttcccttt cttctttagc gacttgatgc
     1621 tcttgatctt ccaatacgca acctaaagta aaatgcccca cagcgctgag tgcatataat
     1681 gcattctcta gtgaaaaacc ttgttggcat aaaaaggcta attgattttc gagagtttca
     1741 tactgttttt ctgtaggccg tgtacctaaa tgtacttttg ctccatcgcg atgacttagt
     1801 aaagcacatc taaaactttt agcgttatta cgtaaaaaat cttgccagct ttccccttct
     1861 aaagggcaaa agtgagtatg gtgcctatct aacatctcaa tggctaaggc gtcgagcaaa
     1921 gcccgcttat tttttacatg ccaatacaat gtaggctgct ctacacctag cttctgggcg
     1981 agtttacggg ttgttaaacc ttcgattccg acctcattaa gcagctctaa tgcgctgtta
     2041 atcactttac ttttatctaa tctggacatc tagtatttct cctctttctc tagtagctag
     2101 cataatccct aggactgagc tagccatcag gttaacaaaa aagcaggaat atccctatca
     2161 gtgatagaca gacatcccta tcagtgatag agtataaagt taactgaaac accgggtctt
     2221 cgagaagacc tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     2281 cttgaaaaag tggcaccgag tcggtgcttt ttttgaagct tgggcccgaa caaaaactca
     2341 tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttaaa
     2401 cggtctccag cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa
     2461 atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt
     2521 cccacctgac cccatgccga actcagaagt gaaacgccgt agcgcgtcgc aggctgggtg
     2581 ccaagctctc gggtaacatc aaggcccgat ccttggagcc cttgccctcc cgcacgatga
     2641 tcgtgccgtg atcgaaatcc agatccttga cccgcagttg caaaccctca ctgatccgca
     2701 tgcccgttcc atacagaagc tgggcgaaca aacgatgctc gccttccaga aaaccgagga
     2761 tgcgaaccac ttcatccggg gtcagcacca ccggcaagcg ccgcgacggc cgaggtcatc
     2821 cgatctcctg aagccagggc agatccgtgc acagcacctt gccgtagaag aactgcaagg
     2881 ccgccaatgc ctgacgatgc gtggagaccg aaaccttgcg ctcgttcgcc agccaggaca
     2941 gaaatgcctc gacttcgctg ctgcccaagg ttgccgggtg acgcacaccg tggaaacgga
     3001 tgaaggcacg aacccagtgg acataagcct gttcggttcg taagctgtaa tgcaagtagc
     3061 gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt aacggcgcag
     3121 tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg cctcgggcat
     3181 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
     3241 ttacgcagca gggcagtcgc cctaaaacaa agttaaacat catgagggaa gcggtgatcg
     3301 ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga
     3361 cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg
     3421 atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga
     3481 tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag
     3541 aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac
     3601 tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga
     3661 tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag
     3721 gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa
     3781 atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag
     3841 tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg
     3901 tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag
     3961 ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg
     4021 aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa taatgtctaa
     4081 caattcgttc aagccgacgc cgcttcgcgg cgcggcttaa ctcaagcgtt agatgcacta
     4141 agcacataat tgctcacagc caaactatca ggtcaagtct gcttttatta tttttaagcg
     4201 tgcataataa gccctacaca aattgggaga tatatcatga aaggctggct ttttcttgtt
     4261 atcgcaatag ttggcgaagt aatcgcaaca tccgcattaa aatctagcga gggctttact
     4321 aagctgatcc ggtggatgac cttttgaatg acctttaata gattatatta ctaattaatt
     4381 ggggacccta gaggtcccct tttttatttt aaaaattttt tcacaaaacg gtttacaagc
     4441 ataaagctct aggaaaataa agtcaaagtg cttctataat ttaagatcta tttttaaaca
     4501 gtacagcttt acagaaagaa aaaccagaag ggacgcctat tttatctcgt tttcttccca
     4561 gagtagcagg acattttttt ctgtaaaagc agagcaattt gtactgaaat cattatcaat
     4621 aatgacatta gattcttcga tcaagacatc tgtaccgttg cttatatctg cttcagagcc
     4681 taatacaatg gggtattgtg gttgttgaca tgcagcatta acgagtactt tgactgggaa
     4741 tacaaccgaa tcatcgctat aggtgtagac attggcgtgg tagtaatagg tatagtcatc
     4801 atagtcgtaa ttatattcat aaccataccg atactggtaa tcaaaatcaa taaatacagg
     4861 ttcacttttt gcctgttgaa caacgataaa tgatagacca ctaagggctg ctgtaaacag
     4921 tttattcatt ttgctttctc catcccttga cttaattaag cttggcgtaa tcatggtcat
     4981 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5041 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5101 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5161 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5221 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5281 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5341 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5401 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5461 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5521 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5581 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5641 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5701 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5761 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5821 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5881 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5941 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6001 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6061 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6121 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6181 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6241 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6301 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6361 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6421 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6481 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6541 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6601 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6661 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6721 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6781 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6841 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6901 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6961 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7021 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7081 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     7141 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc
//
