LOCUS       Exported                8082 bp ds-DNA     circular SYN 29-APR-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8082)
  AUTHORS   Kaiyi Jiang
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Nov 13, 2023 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8082
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          6801..6868
     source          6801..6868
     misc_feature    1..683
                     /label=ori
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    851..990
                     /label=bom
                     /note="basis of mobility region from pBR322"
     misc_feature    1076..1098
                     /label=pGEX_3_primer
     misc_feature    complement(1092..1283)
                     /label=ROP
     CDS             complement(1092..1283)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    complement(1092..1259)
                     /label=ROP
     gene            complement(1922..2536)
                     /label=tet (576 - 1190)
     CDS             complement(2595..3677)
                     /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"
     promoter        complement(3678..3755)
                     /gene="lacI"
                     /label=lacI promoter
     gene            complement(3769..4032)
                     /label=tet (300 - 563)
     primer_bind     4063..4082
                     /label=Kj184
     primer_bind     4064..4083
                     /label=T7
     promoter        4064..4082
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    4082..4109
                     /label=lacO
     protein_bind    4083..4107
                     /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)."
     RBS             4122..4144
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4163..4180
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4190..4207
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4220..4303
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             4304..4597
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     4573..4600
                     /label=PD9801
     CDS             4601..6160
                     /codon_start=1
                     /label=ismimi
                     /translation="MKEAVKNVKPKVPAKKRIITGSKTKKKVFVKKKPPDKKPLKKPVK
                     KTVKTYKLKSIYVSNKDLKMSKWIPTPKKEFTEIETNSWYEHRKFENPNGSPIQSYNKI
                     VPVVPPESIKQQNLANKRKKTNRPIVFISSEKIRIYPTKEQQKILQTWFRLFACMYNSS
                     IDYINSKKVVLESGRINVAATRKVCNKISVRKALKTIRDNLIKSTNPSIMTHIMDEAIG
                     LACSNYKTCLTNYIEGQIKKFDIKPWSISKRRKIIVIEPGYFKGNSFCPTVFPKMKSSK
                     PLIMIDKTVTLQYDSDTRKYILFVPRVTPKYSVNKEKNSCGIDPGLRDFLTVYSENETQ
                     SICPIEIVVNTTKNEYKKIDKINEIIKTKPNLNSKRKKKLNRGLRKYHRRVTNKMKDMH
                     YKVSHELVNTFDKICIGKLNVKSILSKANTVLKSALKRKLATLSFYRFTQRLTHMGYKY
                     GTEVVNVNEYLTTKTCSNCGKIKDLGASKIYECESCGMYADRDENAAKNILKVGLKPWY
                     KQK"
     primer_bind     complement(5559..5595)
                     /label=Primer 5
     CDS             5570..5572
                     /label=catalytic D
     primer_bind     5578..5618
                     /label=Primer 7
     primer_bind     complement(5826..5888)
                     /label=Primer 8
     CDS             5846..5848
                     /label=TNPB Cat E
     primer_bind     5872..5905
                     /label=Primer 6
     primer_bind     complement(5977..6021)
                     /label=KJ395
     CDS             5999..6001
                     /label=Cat E
     primer_bind     6004..6031
                     /label=KJ197
     CDS             6101..6103
                     /label=Cat D
     primer_bind     complement(6164..6181)
                     /label=PD9802
     primer_bind     6164..6181
                     /label=PD9797
     terminator      6224..6271
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6433..6474
                     /label=Kj184
     misc_feature    6433..6450
                     /label=gibson
     promoter        6456..6474
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6475..6499
                     /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)."
     misc_feature    6554..6779
                     /label=fRNA
     misc_feature    6730..6779
                     /label=IRR
     primer_bind     complement(6739..6779)
                     /label=KJ185
     primer_bind     6762..6818
                     /label=KJ579
     misc_feature    6780..6800
                     /label=21nt guide
     ncRNA           6801..6868
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      6869..6916
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(6906..6937)
                     /label=PD9800
     misc_feature    6919..6935
                     /label=gibson
     primer_bind     complement(6983..7007)
                     /label=FP0302
     promoter        7023..7127
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7128..7988
                     /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"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      781 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      841 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
      901 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
      961 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     1021 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     1081 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     1141 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     1201 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     1261 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     1321 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     1381 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     1441 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     1501 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     1561 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     1621 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     1681 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     1741 cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg
     1801 gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag
     1861 aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca
     1921 ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt
     1981 atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa
     2041 tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc
     2101 cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg
     2161 gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg
     2221 tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct
     2281 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     2341 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     2401 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     2461 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     2521 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     2581 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2641 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     2701 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     2761 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     2821 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     2881 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     2941 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     3001 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     3061 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     3121 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     3181 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     3241 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     3301 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     3361 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     3421 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     3481 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     3541 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     3601 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     3661 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     3721 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     3781 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     3841 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     3901 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     3961 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     4021 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     4081 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     4141 gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg
     4201 cggcagccat atggctagct ggagccatcc gcagtttgaa aaaggtggtg gtagcggtgg
     4261 tggttcaggt ggtagtgcat ggtcacaccc tcagtttgag aaaatgtcgg actcagaagt
     4321 caatcaagaa gctaagccag aggtcaagcc agaagtcaag cctgagactc acatcaattt
     4381 aaaggtgtcc gatggatctt cagagatctt cttcaagatc aaaaagacca ctcctttaag
     4441 aaggctgatg gaagcgttcg ctaaaagaca gggtaaggaa atggactcct taagattctt
     4501 gtacgacggt attagaatcc aagctgatca gacccctgaa gatttggaca tggaggataa
     4561 cgatattatt gaggctcaca gagaacagat tggtggatcc atgaaagagg cagttaaaaa
     4621 tgtcaaacca aaagttcccg ctaaaaagag gattattact ggaagtaaaa ctaagaaaaa
     4681 ggtttttgtc aagaagaaac cacctgataa gaaacctctt aagaaacccg tcaaaaaaac
     4741 tgttaaaaca tataaactaa aatctattta tgtttctaat aaagatctta aaatgtccaa
     4801 atggattccc actcctaaaa aagaatttac tgaaattgaa actaacagtt ggtatgaaca
     4861 caggaaattt gagaatccca atggatctcc tatacaatcc tataacaaga ttgtgcctgt
     4921 agttcctccg gaatctatta aacaacagaa tttagcaaac aaacgtaaga aaacaaatag
     4981 acctattgtt tttatttctt ctgagaaaat cagaatttat cccaccaaag agcaacaaaa
     5041 aattttacaa acttggtttc gtttatttgc atgtatgtat aatagttcta ttgattacat
     5101 taacagtaag aaagttgttc ttgaatctgg tagaattaat gtcgctgcaa ccaggaaagt
     5161 ttgtaataaa attagcgtta gaaaagctct aaaaactatt agagacaatt taatcaaatc
     5221 cactaatcca tcaataatga cccatataat ggacgaagcg attggtttag cttgttcaaa
     5281 ttataagaca tgtttaacca attatattga aggtcaaatt aaaaaatttg atattaaacc
     5341 ttggtctatc tctaaaagac gaaagattat tgtcattgaa cctggatact ttaaaggaaa
     5401 ttctttttgt cccacagttt ttcccaaaat gaaatcttct aaaccactta ttatgattga
     5461 taaaactgtg actttgcaat acgattctga tactagaaaa tatatcttat ttgtacctag
     5521 agtcactcct aagtattctg tcaataagga aaaaaatagt tgtggtattg atcctggtct
     5581 tagagatttc cttacggttt attctgaaaa tgaaactcaa tcaatttgtc ctattgaaat
     5641 cgtagttaac accactaaaa atgaatacaa gaaaattgat aagatcaacg aaatcatcaa
     5701 aactaaacca aatcttaata gtaaacgtaa aaagaaactt aatagaggct tacgtaaata
     5761 tcatagaaga gttactaata agatgaaaga catgcactat aaagtttctc atgaattagt
     5821 aaatactttt gataaaattt gtataggtaa actaaatgta aagtctatct tatcgaaagc
     5881 caatactgtt ctaaaaagtg ccttgaaaag aaaacttgca actctgagtt tttataggtt
     5941 tactcagaga ttaactcata tgggttataa gtatggcact gaagtggtta atgtcaatga
     6001 atatttgacc acaaaaacct gctcaaactg cggcaagatt aaagatcttg gagctagcaa
     6061 gatttacgaa tgtgagtctt gtggaatgta cgcagatcgt gatgagaatg ctgccaagaa
     6121 tatcctaaaa gtaggattaa agccttggta caaacaaaaa taggcggccg cactcgaggc
     6181 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     6241 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc
     6301 cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg
     6361 gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag
     6421 caatttaact gtgataaact accgcattaa agctttaata cgactcacta taggggaatt
     6481 gtgagcggat aacaattcca tgagaatgct gccaagaata tcctaaaagt aggattaaag
     6541 ccttggtaca aacaaaaata gtctaataaa atcaggggta cattccgcta gtactccacc
     6601 ctacgggtta agcaaatgag aatatcgaaa cggtatgcac aggattcttc gagtgataat
     6661 cttaggatga ctcactaagg agatgactaa agtgtatcat tcaatattgt attgaacggt
     6721 attcttccat agagagttga tttttggagt atccagaaat atcaactttt tatgagcggg
     6781 cagccacctc cttgttattg ggccggcatg gtcccagcct cctcgctggc gccggctggg
     6841 caacatgctt cggcatggcg aatgggacct agcataaccc cttggggcct ctaaacgggt
     6901 cttgaggggt tttttggaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     6961 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     7021 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     7081 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     7141 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     7201 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     7261 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     7321 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     7381 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     7441 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     7501 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     7561 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     7621 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     7681 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     7741 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     7801 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     7861 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     7921 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     7981 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     8041 tttaatttaa aaggatctag gtgaagatcc tttttgataa tc
//
