LOCUS       Exported                6746 bp ds-DNA     circular SYN 13-APR-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6746)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Jun 28, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6746
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     2..21
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             109..131
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             151..180
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             190..1290
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     646..665
                     /label=HS_2
     primer_bind     1264..1287
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     primer_bind     complement(1332..1361)
                     /label=HS_85
     CDS             1345..1365
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     1347..1394
                     /label=oBA2469_ECOR31HamA_purification_F
     misc_feature    1347..1371
                     /label=Gibson_UP
     misc_feature    1372..2151
                     /label=ECOR31_Hachiman
     CDS             1372..2151
                     /codon_start=1
                     /label=HamA (SAVED?)
                     /translation="MANFEDWCDSTERNISDHYLQSITARDAECMFGVQVMAALIPEHY
                     ASPRNIANAFEALGKPGLAAYIAGKLPETKQIRSGDLGEIFATEWINARSNGYKTPIKR
                     LRWKDHRNMSMRGEDVIGIYIDQSSQQLFFLKTEAKSRAKMTGEVVSEARDNLNKEQGL
                     PSSHALMFIADRLNEQGEELLAKAILNATLRQGIVPGCVRHLIFLLSGNSSETMLTTSI
                     EKYTGQNNQWGVCLRIARHGEFIAATFEKVISDASNS"
     primer_bind     1372..1394
                     /label=oBA2343_ECOR31_Hachiman_Gib_p1_F
     primer_bind     complement(2132..2178)
                     /label=oBA2470_ECOR31HamA_purification_R
     misc_feature    2152..2178
                     /label=Gibson_DN
     primer_bind     complement(2277..2295)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2291..2338
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(2704..2732)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2759..2777)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2845..2949
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2950..3810
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3168..3187)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3981..4569
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4470..4489
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4572..4581
                     /label=HS_1
     primer_bind     4723..4740
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4755..4894
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4820..4839)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4980..5002
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4996..5187)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6645..6664
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6657..6674
                     /label=HS_1
ORIGIN
        1 ttaatacgac tcactatagg gagaccacaa cggtttccct ctagtgccgg ctccggagag
       61 ctctttaatt aagcggccgc cctgcaggac tcgagttcta gaaataattt tgtttaactt
      121 taagaaggag atatacatat gaaatcttct caccatcacc atcaccatca ccatcaccat
      181 ggttcttcta tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc
      241 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc
      301 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc
      361 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg
      421 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc
      481 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt
      541 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat
      601 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc
      661 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac
      721 gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt
      781 gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc
      841 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac
      901 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa
      961 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca
     1021 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac
     1081 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt
     1141 attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg
     1201 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact
     1261 gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacaataac
     1321 aataacaaca acctcgggat cgaggaaaac ctgtacttcc aatccaatgc aatggcaaat
     1381 ttcgaggact ggtgtgattc gacagagcgt aatatcagcg accattattt gcagtcaata
     1441 acggccaggg atgccgagtg tatgttcggc gttcaggtta tggccgcgct aatacctgaa
     1501 cactatgcgt caccaagaaa tatcgctaac gcatttgaag ctctaggtaa accggggctg
     1561 gcggcataca ttgctggaaa attgcctgaa acgaaacaga ttcgttccgg tgatttgggt
     1621 gagatatttg caactgaatg gataaatgca cgtagcaatg gctataaaac gcctataaaa
     1681 agattgcgct ggaaagatca tcgcaatatg tcgatgcgtg gtgaggacgt cattggtatt
     1741 tacatagatc agtcttctca acaacttttt ttccttaaaa ccgaagcgaa aagtcgcgcc
     1801 aaaatgacgg gagaggtggt ttctgaagcc cgcgataact tgaataaaga gcaagggctg
     1861 ccatcttcac atgccctaat gttcatagca gatcgcctga atgaacaggg tgaggagctt
     1921 ttggctaaag cgatcttaaa tgcaacgctt cggcaaggaa ttgttccagg atgcgtccgg
     1981 catcttatct ttctattatc gggcaactct tcagaaacta tgctgaccac ttcaattgaa
     2041 aagtatacag gtcaaaataa tcagtggggt gtttgtctaa gaattgctcg ccatggtgag
     2101 tttattgccg caacttttga aaaggtgatt agcgatgcca gcaacagcta ataacattgg
     2161 aagtggataa cggatccgcg atcgcggcgc gccacctggt ggccggccgg taccacgcgt
     2221 gcgcgctgat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     2281 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     2341 gaaaggagga actatatccg gatatccaca ggacgggtgt ggtcgccatg atcgcgtagt
     2401 cgatagtggc tccaagtagc gaagcgagca ggactgggcg gcggccaaag cggtcggaca
     2461 gtgctccgag aacgggtgcg catagaaatt gcatcaacgc atatagcgct agcagcacgc
     2521 catagtgact ggcgatgctg tcggaatgga cgatatcccg caagaggccc ggcagtaccg
     2581 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
     2641 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
     2701 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
     2761 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     2821 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2881 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata acattgaaaa
     2941 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3001 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3061 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3121 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3181 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3241 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3301 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3361 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3421 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3481 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3541 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3601 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3661 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3721 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3781 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3841 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3901 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3961 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4021 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4081 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4141 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4201 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4261 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4321 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4381 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4441 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4501 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4561 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4621 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4681 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4741 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4801 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4861 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4921 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4981 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5041 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     5101 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5161 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5221 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5281 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5341 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5401 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5461 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5521 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5581 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5641 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga
     5701 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg
     5761 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct
     5821 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca
     5881 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca
     5941 taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc
     6001 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac
     6061 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct
     6121 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     6181 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     6241 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     6301 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     6361 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     6421 gggttgaagg ctctcaaggg catcggtcga cgctctccct tatgcgactc ctgcattagg
     6481 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     6541 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     6601 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     6661 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     6721 aggatcgaga tctcgatccc gcgaaa
//
