LOCUS       Exported                7266 bp ds-DNA     circular SYN 02-AUG-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7266)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Jun 6, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7266
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             910..951
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    988..2028
                     /label=BoNT N-terminus
     misc_feature    2059..2475
                     /label=hLOV
     primer_bind     2457..2502
                     /label=10GS-BoNTC-FP
     primer_bind     complement(2457..2478)
                     /label=5GS-RP
     primer_bind     complement(2457..2478)
                     /label=2GS-RP
     primer_bind     complement(2457..2478)
                     /label=10GS-RP
     primer_bind     2460..2502
                     /label=2GS-BoNTC-FP
     primer_bind     2464..2502
                     /label=5GS-BoNTC-FP
     primer_bind     2477..2502
                     /label=10GS-BoNTC-FP original
     misc_feature    2479..2784
                     /label=BoNT C-terminus
     promoter        complement(2818..2836)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     polyA_signal    2862..3086
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3132..3560
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        3574..3904
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3755..3890
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3971..4765
                     /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"
     polyA_signal    4939..5060
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5109..5125)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5133..5149
                     /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(5157..5187)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5202..5223
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5511..6099)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6270..7130)
                     /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"
     promoter        complement(7131..7235)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg gcaagcccat ccccaacccc ctgctgggcc tggacagcac caagggctcg
      961 ggctcgacct cgggctcggg caccggtccc ttcgtgaaca agcagttcaa ctacaaggat
     1021 cccgtgaacg gcgtggacat tgcctacatc aagatcccca acgccggcca gatgcagccc
     1081 gtgaaggcct ttaagatcca caacaagatc tgggtcatcc ccgagcgcga taccttcacc
     1141 aaccccgagg aaggcgatct gaacccccca cccgaagcca agcaggtccc cgtgtcctac
     1201 tacgatagca cctacctgag caccgataac gagaaggata actacctgaa gggcgtgacc
     1261 aagctgttcg agcgcatcta cagcaccgat ctgggccgca tgctgctgac ctcgatcgtg
     1321 cgcggcatcc cgttctgggg cggcagcacc atcgataccg agctgaaggt catcgatacc
     1381 aactgcatca acgtgatcca gcccgatggc agctaccgca gcgaggaact gaacctggtc
     1441 atcatcggcc ccagcgccga tatcatccag ttcgagtgca agagcttcgg ccacgaggtg
     1501 ctgaacctga cccgcaacgg ctacggcagc acccagtaca tccgcttcag ccccgatttc
     1561 accttcggct tcgaggaatc cctggaagtg gataccaacc ccctgctggg agccggcaag
     1621 ttcgccaccg atccagccgt gaccctggcc cacgagctga ttcatgccgg ccaccgcctg
     1681 tacggcattg ccatcaaccc caaccgcgtg ttcaaggtca acaccaacgc ctactacgag
     1741 atgagcggcc tggaagtgtc cttcgaggaa ctgcgcacct tcggcggaca cgatgccaag
     1801 ttcatcgata gcctgcagga aaacgagttc cgcctgtact actacaacaa gttcaaggat
     1861 atcgccagca ccctgaacaa ggcgaagagc atcgtgggca ccaccgccag cctgcagtac
     1921 atgaagaatg tgttcaagga aaagtacctg ctgagcgagg ataccagcgg caagttctcc
     1981 gtggataagc tgaagttcga caagctgtac aagatgctga ccgagatcgg gagcggcagc
     2041 ggggggtccg gtagcgggga gtttcgggca accacactgg aacggatcga gaaaagcttc
     2101 gtgattactg atccgagact gcctgacaac ccaatcattt ttgtgagcga ttccttcctg
     2161 cagctgacag aatattctcg ggaagagatc ctggggcgca attgccgttt tctgcaggga
     2221 cccgagacag accgtgccac tgttcggaaa atcagagatg ctattgacaa ccagactgaa
     2281 gtgaccgttc agctgatcaa ttataccaag agcggcaaga agttctggaa cgtgttccac
     2341 ctgcagccga tgcgcgatta taagggcgac gtccagtact tcattggcgt gcagctggat
     2401 ggcaccgaac gtcttcatgg cgccgctgag cgtgaggcgg tctgcctggt caaaaagaca
     2461 gcctttcaga ttgctggcta caccgaggac aacttcgtga agttcttcaa ggtgctgaat
     2521 cgcaagacct acctgaactt cgataaggcc gtgttcaaga ttaacatcgt gcccaaggtc
     2581 aactacacca tctacgatgg cttcaacctg cgcaacacca acctggccgc caacttcaac
     2641 ggccagaaca ccgagatcaa caacatgaac ttcaccaagc tgaagaattt caccggcctg
     2701 ttcgagttct acaagctgct gtgcgtgcgc ggaatcatca ccagcaagac caagagcctg
     2761 gataagggct acaacaaggc caagtaataa ctcgagcatg catctagagg gccctattct
     2821 atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     2881 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     2941 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3001 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3061 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     3121 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3181 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3241 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcggggca tccctttagg
     3301 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3361 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3421 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3481 ttttgattta taagggattt tggggatttc ggcctattgg ttaaaaaatg agctgattta
     3541 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3601 ccaggctccc caggcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3661 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3721 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3781 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3841 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3901 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     3961 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     4021 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4081 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4141 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4201 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4261 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     4321 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     4381 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     4441 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     4501 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     4561 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4621 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4681 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     4741 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     4801 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     4861 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     4921 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4981 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     5041 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     5101 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5161 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5221 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5281 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5341 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5401 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5461 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5521 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5581 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5641 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5701 tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5761 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5821 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5881 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5941 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6001 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6061 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6121 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6181 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6241 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6301 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6361 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6421 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6481 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6541 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6601 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6661 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6721 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6781 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6841 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6901 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6961 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7021 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7081 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7141 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7201 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7261 gacgtc
//
