LOCUS       WT_His_POLI_(pJM        7631 bp DNA     circular SYN 07-APR-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7631)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 20, 2023 from SnapGene 6.2.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7631
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             17..39
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             46..111
                     /codon_start=1
                     /label=tag
                     /translation="MGSSHHHHHHSSGLEVLFQGPH"
     CDS             58..75
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             85..108
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             112..2259
                     /codon_start=1
                     /label=WT POLI CDS
                     /translation="MELADVGAAASSQGVHDQVLPTPNASSRVIVHVDLDCFYAQVEMI
                     SNPELKDKPLGVQQKYLVVTCNYEARKLGVKKLMNVRDAKEKCPQLVLVNGEDLTRYRE
                     MSYKVTELLEEFSPVVERLGFDENFVDLTEMVEKRLQQLQSDELSAVTVSGHVYNNQSI
                     NLLDVLHIRLLVGSQIAAEMREAMYNQLGLTGCAGVASNKLLAKLVSGVFKPNQQTVLL
                     PESCQHLIHSLNHIKEIPGIGYKTAKCLEALGINSVRDLQTFSPKILEKELGISVAQRI
                     QKLSFGEDNSPVILSGPPQSFSEEDSFKKCSSEVEAKNKIEELLASLLNRVCQDGRKPH
                     TVRLIIRRYSSEKHYGRESRQCPIPSHVIQKLGTGNYDVMTPMVDILMKLFRNMVNVKM
                     PFHLTLLSVCFCNLKALNTAKKGLIDYYLMPSLSTTSRSGKHSFKMKDTHMEDFPKDKE
                     TNRDFLPSGRIESTRTRESPLDTTNFSKEKDINEFPLCSLPEGVDQEVFKQLPVDIQEE
                     ILSGKSREKFQGKGSVSCPLHASRGVLSFFSKKQMQDIPINPRDHLSSSKQVSSVSPCE
                     PGTSGFNSSSSSYMSSQKDYSYYLDNRLKDERISQGPKEPQGFHFTNSNPAVSAFHSFP
                     NLQSEQLFSRNHTTDSHKQTVATDSHEGLTENREPDSVDEKITFPSDIDPQVFYELPEA
                     VQKELLAEWKRTGSDFHIGHK"
     CDS             184..777
                     /codon_start=1
                     /label=Fingers-palm fragment
                     /translation="ASSRVIVHVDLDCFYAQVEMISNPELKDKPLGVQQKYLVVTCNYE
                     ARKLGVKKLMNVRDAKEKCPQLVLVNGEDLTRYREMSYKVTELLEEFSPVVERLGFDEN
                     FVDLTEMVEKRLQQLQSDELSAVTVSGHVYNNQSINLLDVLHIRLLVGSQIAAEMREAM
                     YNQLGLTGCAGVASNKLLAKLVSGVFKPNQQTVLL"
     CDS             766..990
                     /codon_start=1
                     /label=Thumb domain
                     /translation="TVLLPESCQHLIHSLNHIKEIPGIGYKTAKCLEALGINSVRDLQT
                     FSPKILEKELGISVAQRIQKLSFGEDNSPV"
     CDS             997..1371
                     /codon_start=1
                     /label=Little Finger Domain
                     /translation="SGPPQSFSEEDSFKKCSSEVEAKNKIEELLASLLNRVCQDGRKPH
                     TVRLIIRRYSSEKHYGRESRQCPIPSHVIQKLGTGNYDVMTPMVDILMKLFRNMVNVKM
                     PFHLTLLSVCFCNLKALNTAK"
     misc_feature    1429..1449
                     /label=KKK440/444/446AAA
     misc_feature    1429..1431
                     /label=GCG
     misc_RNA        1441..1443
                     /label=GCG
     misc_difference 1447..1449
                     /label=GCG
     misc_feature    1849..1851
                     /label=S580A_GCG
     CDS             2297..2314
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2381..2428
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2465..2823
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2838..2854)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2862..2878
                     /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(2886..2916)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2931..2952
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(3480..4430)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      4478..4700
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(5124..5939)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        6348..6425
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             6426..7508
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    7573..7589
                     /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)."
     protein_bind    7606..7622
                     /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)."
ORIGIN
        1 gaattctaga aataattttg tttaacttta agaaggagat ataacatggg cagcagccat
       61 catcatcatc atcacagcag cggcctggaa gttctgttcc aggggcccca tatggaactg
      121 gcggatgttg gcgccgcggc gagcagccag ggcgttcatg atcaggtgtt accaaccccg
      181 aacgcgagca gccgtgtgat tgtgcatgtg gatctggatt gcttttatgc gcaggtggaa
      241 atgattagca acccggaact gaaagataaa ccgctgggcg tgcagcagaa atatctggtg
      301 gtgacctgca actacgaggc ccgtaaactg ggcgtgaaaa aactgatgaa cgtgcgtgat
      361 gcgaaagaaa aatgcccgca gctggtgctg gtgaacggcg aagatctgac ccgttatcgt
      421 gaaatgagct ataaagtgac cgaactgctg gaagaattta gcccggtggt ggaacgtctg
      481 ggctttgatg aaaactttgt ggatctgacc gaaatggtgg aaaaacgtct gcagcagctg
      541 cagagcgatg aactgagcgc ggtgaccgtg agcggccatg tgtataacaa ccagagcatt
      601 aacctgctgg atgtgctgca tattcgtctg ctggtgggca gccagattgc ggcggaaatg
      661 cgtgaagcga tgtataacca gctgggcctg accggctgcg cgggcgtggc gagcaacaaa
      721 ctgctggcga aactggtgag cggcgtgttt aaaccgaacc agcagaccgt gctgctgccg
      781 gaaagctgcc agcatctgat tcatagcctg aaccatatta aagaaattcc gggcattggc
      841 tataaaaccg cgaaatgcct ggaagcgctg ggcattaaca gcgtgcgtga tctgcagacc
      901 tttagcccga aaattctgga aaaagaactg ggcattagcg tggcgcagcg tattcagaaa
      961 ctgagctttg gcgaagataa cagcccggtg attctgagcg gtccgccaca aagctttagc
     1021 gaagaagata gctttaaaaa atgcagcagc gaagtggaag cgaaaaacaa aattgaggaa
     1081 ttactggcca gcctgctgaa ccgtgtgtgc caggatggcc gtaaaccgca taccgtgcgt
     1141 ctgattattc gtcgttatag cagcgaaaaa cattatggcc gtgaaagccg tcagtgcccg
     1201 attccgagcc atgtgattca gaaactgggc accggcaact atgatgtgat gaccccgatg
     1261 gtggatattc tgatgaaact gtttcgtaac atggtgaacg tgaaaatgcc gtttcatctg
     1321 accctgctga gcgtgtgctt ttgcaacctg aaagcgctga acaccgcgaa aaaaggcctg
     1381 attgattatt atctgatgcc gagcctgagc accaccagcc gtagcggcaa acatagcttt
     1441 aaaatgaaag atacccacat ggaagatttt ccgaaagata aagaaaccaa ccgtgatttt
     1501 ctgccgagcg gccgtattga aagcacccgt acccgtgaaa gcccgctgga taccaccaac
     1561 tttagcaaag aaaaagatat taacgagttt ccgttatgct ctctgccgga aggcgtggat
     1621 caggaagtgt ttaaacagct gccggttgat atccaggaag aaattctgag cggcaaaagc
     1681 cgtgaaaaat ttcagggcaa aggcagcgtg agctgcccgc tgcatgcgag ccgtggcgtg
     1741 ctgagctttt ttagcaaaaa acagatgcag gatattccga ttaacccgcg tgatcatctg
     1801 agcagcagca aacaggtgag cagcgtgagc ccgtgcgaac cgggcaccag cggctttaac
     1861 agcagcagca gcagctatat gagcagccag aaagattata gctattatct ggataaccgt
     1921 ctgaaagatg aacgtattag ccagggcccg aaagaaccgc agggctttca ttttaccaac
     1981 agcaacccgg cggtgagcgc cttccacagc tttccgaacc tgcagagcga acagctgttt
     2041 agccgtaacc atacgaccga tagccataaa cagaccgtgg cgaccgatag ccatgaaggc
     2101 ctgaccgaaa accgtgaacc ggatagcgtg gatgaaaaaa ttacctttcc gagcgatatt
     2161 gatccgcagg tgttttatga actgccggaa gcggtgcaga aagaactgct ggcggaatgg
     2221 aaacgtaccg gcagcgattt tcatattggc cataaataag tcggcctgcg aattcgtcga
     2281 cgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     2341 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     2401 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     2461 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2521 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2581 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2641 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2701 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2761 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2821 ttaaattcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2881 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2941 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3001 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctat
     3061 ttcttccaga attgccatga ttttttcccc acgggaggcg tcactggctc ccgtgttgtc
     3121 ggcagctttg attcgataag cagcatcgcc tgtttcaggc tgtctatgtg tgactgttga
     3181 gctgtaacaa gttgtctcag gtgttcaatt tcatgttcta gttgctttgt tttactggtt
     3241 tcacctgttc tattaggtgt tacatgctgt tcatctgtta cattgtcgat ctgttcatgg
     3301 tgaacagctt taaatgcacc aaaaactcgt aaaagctctg atgtatctat cttttttaca
     3361 ccgttttcat ctgtgcatat atggacagtt ttccctttga tatctaacgg tgaacagttg
     3421 ttctactttt gtttgttagt cttgatgctt cactgataga tacaagagcc ataagaacct
     3481 cagatccttc cgtatttagc cagtatgttc tctagtgtgg ttcgttgttt ttgcgtgagc
     3541 catgagaacg aaccattgag atcatgctta ctttgcatgt cactcaaaaa ttttgcctca
     3601 aaactggtga gctgaatttt tgcagttaaa gcatcgtgta gtgtttttct tagtccgtta
     3661 cgtaggtagg aatctgatgt aatggttgtt ggtattttgt caccattcat ttttatctgg
     3721 ttgttctcaa gttcggttac gagatccatt tgtctatcta gttcaacttg gaaaatcaac
     3781 gtatcagtcg ggcggcctcg cttatcaacc accaatttca tattgctgta agtgtttaaa
     3841 tctttactta ttggtttcaa aacccattgg ttaagccttt taaactcatg gtagttattt
     3901 tcaagcatta acatgaactt aaattcatca aggctaatct ctatatttgc cttgtgagtt
     3961 ttcttttgtg ttagttcttt taataaccac tcataaatcc tcatagagta tttgttttca
     4021 aaagacttaa catgttccag attatatttt atgaattttt ttaactggaa aagataaggc
     4081 aatatctctt cactaaaaac taattctaat ttttcgcttg agaacttggc atagtttgtc
     4141 cactggaaaa tctcaaagcc tttaaccaaa ggattcctga tttccacagt tctcgtcatc
     4201 agctctctgg ttgctttagc taatacacca taagcatttt ccctactgat gttcatcatc
     4261 tgagcgtatt ggttataagt gaacgatacc gtccgttctt tccttgtagg gttttcaatc
     4321 gtggggttga gtagtgccac acagcataaa attagcttgg tttcatgctc cgttaagtca
     4381 tagcgactaa tcgctagttc atttgctttg aaaacaacta attcagacat acatctcaat
     4441 tggtctaggt gattttaatc actataccaa ttgagatggg ctagtcaatg ataattacta
     4501 gtccttttcc tttgagttgt gggtatctgt aaattctgct agacctttgc tggaaaactt
     4561 gtaaattctg ctagaccctc tgtaaattcc gctagacctt tgtgtgtttt ttttgtttat
     4621 attcaagtgg ttataattta tagaataaag aaagaataaa aaaagataaa aagaatagat
     4681 cccagccctg tgtataactc actactttag tcagttccgc agtattacaa aaggatgtcg
     4741 caaacgctgt ttgctcctct acaaaacaga ccttaaaacc ctaaaggctt aagtagcacc
     4801 ctcgcaagct cgggcaaatc gctgaatatt ccttttgtct ccgaccatca ggcacctgag
     4861 tcgctgtctt tttcgtgaca ttcagttcgc tgcgctcacg gctctggcag tgaatggggg
     4921 taaatggcac tacataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4981 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca
     5041 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5101 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     5161 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     5221 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     5281 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     5341 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     5401 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     5461 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     5521 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     5581 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     5641 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     5701 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     5761 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     5821 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     5881 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     5941 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     6001 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6061 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6121 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6181 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     6241 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtgcaggt
     6301 cgactcgaca attcgcgcgc gaaggcgaag cggcatgcat ttacgttgac accatcgaat
     6361 ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     6421 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     6481 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     6541 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     6601 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     6661 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     6721 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     6781 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     6841 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     6901 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     6961 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     7021 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     7081 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     7141 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     7201 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     7261 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     7321 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     7381 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     7441 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     7501 ggcagtgagc gcaacgcaat taatgtaagt tagcgcgaat tgtcgagaaa atttatcaaa
     7561 aagagtgttg acttgtgagc ggataacaat gatacttaga ttcaattgtg agcggataac
     7621 aatttcacac a
//
