LOCUS       Exported                7712 bp ds-DNA     circular SYN 23-JUN-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7712)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Jun 25, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7712
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          402..468
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          5379..5472
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     379..395
                     /label=M13 fwd
     misc_feature    402..418
                     /label=T7 promoter
     misc_feature    419..459
                     /label=HBA1-derived 5UTR
                     /note="CleanCap AG is optimized for production of mRNA that
                     initiates with AGG, conventional transcripts start with 
                     5'-GGG.
                     
                     
                     Best T7 promoter sequence would be 
                     5'TAATACGACTCACTATAAGGNNNNNNNNNNNN 3'  Using that sequence 
                     with the CleanCap AG reagent, N-7113, you will get 95-98% 
                     capped mRNA with the natural CAP1 structure
                     
                     
                     https://www.tebu-bio.com/blog/tips-to-modify-mrna-for-in-vi
                     vo-applications-and-delivery-optimisation/"
     misc_feature    460..468
                     /label=kozak
     CDS             469..489
                     /codon_start=1
                     /label=NLS
                     /translation="PKKKRKV"
     CDS             496..5274
                     /codon_start=1
                     /label=R2Tg_opt
                     /translation="MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKS
                     SDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDASCP
                     KPGPPVSAGAMSLESGLTTHSVLAIERGPNSLANSGSDFGGGGLGLPLRLLRVSVGTQT
                     SRSDWVDLVSWSHPGPTSKSQQVDLVSLFPKHRVDLLSKNDQVDLVAQFLPSKFPPNLA
                     ENDLALLVNLEFYRSDLHVYECVHFAAHWEGLSGLPEVYEQLAPQPCVGETLHSSLPRD
                     SELFVPEEGSSEKESEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNPPCPCCGTRVNS
                     VLNLIEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCRGPETEKAPAGEWICEVC
                     NRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETSNRGAHKRCWTKEEEELLIRLEAQ
                     FEGNKNINKLIAEHITTKTAKQISDKRRLLSRKPAEEPREEPGTCHHTRRAAASLRTEP
                     EMSHHAQAEDRDNGGGMPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKD
                     KSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDGG
                     PGRRPLPGRAAAGGRTMDEIRRHPDKGNGQQRPTKQKSEEQLQAYYKKTLEERLSAGAL
                     NTFPRAFKQVMEGRDIKLVINQTAQDCFGCLESISQIRTATRDKKDTVTREKHPKKPFQ
                     KWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVFKTRWETTG
                     SFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGPDGITLGDVVKMDPEFS
                     RTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLKDINNWRPITIGSILLRLFSRIV
                     TARLSKACPLNPRQRGFIRAAGCSENLKLLQTIIWSAKREHRPLGVVFVDIAKAFDTVS
                     HQHIIHVLQQREVDPHIVGLVSNMYENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLL
                     FNLAMDPLLCKLEESGKGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLT
                     GLKTQGQKCHGFYIKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLRFDPWIGIA
                     RSGLSTKLDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIR
                     TAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMKCFM
                     EKEKMEQLHKKLWIQARGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQKDKFPKPCN
                     WRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRKLLTALQLRANVYPTREF
                     LARGRQDQYIKACRHCDADIESCAHIIGNCPVTQDARIKRHNYICELLLEEAKKKDWVV
                     FKEPHIRDSNKELYKPDLIFVKDARALVVDVTVRYEAAKSSLEEAAAEKVRKYKHLETE
                     VRHLTNAKDVTFVGFPLGARGKWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDI
                     VHMFASRARKSMVM"
     CDS             5281..5301
                     /codon_start=1
                     /label=NLS
                     /translation="PKKKRKV"
     CDS             5308..5373
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    5379..5472
                     /label=alpha-globin-3UTR
     primer_bind     complement(5491..5507)
                     /label=M13 rev
     protein_bind    5515..5531
                     /label=lac operator
     promoter        complement(5539..5569)
                     /label=lac promoter
     rep_origin      complement(5893..6481)
                     /direction=LEFT
                     /label=ori
     CDS             complement(6652..7512)
                     /codon_start=1
                     /label=AmpR
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7513..7617)
                     /label=AmpR promoter
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 ccagtgaatt ctaatacgac tcactataag
      421 gaaataagag agaaaagaag agtaagaaga aatataagag ccaccatgcc caagaagaag
      481 aggaaggttc ccgggatgcc taagagaaag gtgtccagcg ccgagggcgc cgccaaagaa
      541 gagcctaagc ggagaagcgc cagactgagc gccaagcctc cagctaaagt ggaagccaag
      601 cccaagaagg ccgctgccaa ggacaagagc tctgataaga aagtccagac caagggaaaa
      661 cggggcgcta aaggcaagca ggccgaggtg gccaaccagg agacaaagga agatctgcct
      721 gctgagaacg gcgaaaccaa gaccgaggaa agccccgcca gcgacgaggc cggcgagaag
      781 gaggccaagt ctgacgccag ctgccccaag ccaggcccac ctgtgtctgc cggcgccatg
      841 agcctggaat ctggactgac aacacacagc gtgctggcca tcgagagagg acccaacagc
      901 ctggccaatt ccggctctga tttcggcggc ggcggcctgg gcctgccact aagactgctg
      961 cgtgtatcag tgggcacgca gaccagcaga agcgattggg tggatctggt gagctggtcc
     1021 caccctggcc ctacaagcaa gtcccagcag gttgaccttg tgagcctgtt cccgaagcac
     1081 cgggtggacc tgctatccaa aaatgatcag gtggatctgg tcgcgcaatt cctgccttca
     1141 aagttccctc ccaacctggc tgaaaatgac ctcgccctgc tggtgaacct ggaattttac
     1201 agaagcgacc tgcacgtgta cgaatgcgtg cacttcgctg cccattggga gggcctgagc
     1261 ggactgccag aagtctacga acagctcgcc cctcagcctt gcgtgggaga gaccctgcat
     1321 agctccctgc ctcgggatag cgagctgttc gtgcctgagg agggctctag cgaaaaggaa
     1381 tcggaagatg cccccaagac cagccccccc acccctggca aacatggcct ggagcagaca
     1441 ggcgaggaga aggtgatggt taccgtcccc gacaagaacc ctccctgtcc ttgctgcggc
     1501 accagagtga acagcgtgct gaatctgatc gagcacctga aggtgagcca cggcaagcgt
     1561 ggagtttgtt tccggtgcgc caagtgcggc aaggaaaaca gcaactacca cagcgtggtg
     1621 tgccactttc ctaagtgtag aggccctgag acagaaaagg cccctgccgg agaatggatt
     1681 tgtgaggtgt gcaaccggga ttttaccacc aagatcggcc tgggccagca caaacggctg
     1741 gcccaccccg ccgtgagaaa ccaggagagg atcgtggcca gccaacctaa agagacaagc
     1801 aacagaggcg cccacaagcg gtgctggacg aaggaagagg aagaattgtt gatcagactg
     1861 gaagcccagt tcgagggaaa caagaacatc aacaagctga ttgccgagca catcaccacc
     1921 aagaccgcca agcagatcag cgacaagaga aggctgttaa gcagaaagcc tgccgaggag
     1981 cccagagagg aacctggcac ctgccaccac acccggagag ccgccgcttc actgcggacc
     2041 gagcccgaga tgtctcacca cgcccaggcc gaggacagag ataacggcgg aggaatgcct
     2101 aagagaaagg tgtccagcgc cgagggcgcc gccaaagaag agcctaagcg gagaagcgcc
     2161 agactgagcg ccaagcctcc agctaaagtg gaagccaagc ccaagaaggc cgctgccaag
     2221 gacaagagct ctgataagaa agtccagacc aagggaaaac ggggcgctaa aggcaagcag
     2281 gccgaggtgg ccaaccagga gacaaaggaa gatctgcctg ctgagaacgg cgaaaccaag
     2341 accgaggaaa gccccgccag cgacgaggcc ggcgagaagg aggccaagtc tgacggagga
     2401 cccggcagac ggcctctccc cggaagggct gccgccggcg gaagaaccat ggatgagatc
     2461 agaagacacc ccgacaaagg aaatggccag cagcggccta ccaagcagaa atccgaagaa
     2521 cagctgcagg cttactacaa gaagaccctg gaagagagac tgagcgccgg agccctgaac
     2581 acctttccta gagccttcaa acaggtgatg gaaggccggg acataaagct ggtcatcaac
     2641 cagacagccc aggattgctt cggctgtctg gagagcatct cccagattag gaccgctaca
     2701 cgggataaga aggataccgt gacaagagag aaacacccta agaagccctt ccagaagtgg
     2761 atgaaagaca gagcaatcaa gaaaggcaat tacctgagat tccagagact gttttacctg
     2821 gacagaggca agctggccaa aatcattctg gacgacatcg agtgtctgag ttgcgacatc
     2881 cctctgtccg agatctacag cgtgttcaaa actcggtggg aaacaacagg cagcttcaag
     2941 tctctgggcg acttcaagac atacggcaaa gccgataaca cggcttttag agagctgatc
     3001 accgccaagg agattgaaaa gaacgtgcag gagatgagca agggcagcgc ccctggcccc
     3061 gatggcatca ccctgggaga tgtggtgaag atggaccctg agttcagcag aacgatggag
     3121 attttcaatc tgtggctgac caccggcaag attcccgaca tggtgcgggg atgtcggacc
     3181 gtgctgatcc caaagagctc caagcccgat agactgaagg acatcaacaa ctggagaccc
     3241 atcaccatcg gcagcatcct actcagactg tttagcagaa tcgtgaccgc cagactgtcc
     3301 aaggcttgtc ctctgaatcc taggcagaga ggcttcatcc gggctgctgg ttgcagcgag
     3361 aacctgaagc tgttgcagac catcatctgg agcgccaaga gagagcacag acctctgggc
     3421 gtggtgtttg tggacatcgc caaagccttc gacaccgtgt cccaccagca tatcatccac
     3481 gtgctgcaac aaagagaggt ggacccccac atcgtgggcc tggtcagcaa catgtacgag
     3541 aacatcagca cctacatcac aaccaagcgg aacacccaca ccgacaagat ccagatccgg
     3601 gtgggcgtga agcagggcga tcctatgtcc cctcttctgt tcaacctggc catggacccc
     3661 ctgttgtgca agctggagga gagcggcaag ggctaccacc ggggccagag cagcatcaca
     3721 gccatggcgt tcgccgacga tttggtgttg ctgagcgatt cttgggagaa catgaacacc
     3781 aacatcagca tcctggagac cttctgcaac ctgaccggcc tgaaaaccca gggccagaag
     3841 tgccacggct tctatatcaa gcctacaaaa gacagctaca ccatcaatga ctgcgccgcc
     3901 tggaccatca acggcacacc tctgaatatg attgaccctg gcgaaagcga gaagtacctg
     3961 ggcttgagat tcgacccttg gatcggaatc gccagaagcg gcctgagcac caagctcgac
     4021 ttctggttgc agagaatcga ccaggcacca ctgaagcccc tgcaaaagac agacatcctg
     4081 aagacataca ccatccctcg cctaatctat atcgccgacc actctgaggt gaaaaccgcc
     4141 ctgctggaaa ccctggacca aaagatccgg acagccgtga aggaatggct gcacctgccg
     4201 ccttgtacat gcgacgctat cctgtacagc agcaccagag atggcgggct gggcatcact
     4261 aagctggccg gcctcattcc tagcgtgcag gccagacggc tgcatagaat cgcacaaagc
     4321 tctgatgaca ccatgaagtg ctttatggag aaagagaaaa tggaacagct gcacaagaag
     4381 ctgtggattc aggccagagg agaccgagaa aacatcccat ctatctggga ggctcctcct
     4441 agctctgagc ctcctaacaa cgtgtctaca aacagcgagt gggaggcccc tacccagaag
     4501 gacaagttcc ccaagccatg taactggcgg aaaaacgagt tcaagaagtg gaccaagctg
     4561 gccagccagg gccggggcat tgtgaacttc gagcgggaca agatctctaa ccactggatt
     4621 cagtactata gaagaatccc tcaccggaag ctgctgaccg ccctgcagct gagagctaat
     4681 gtatacccca caagagagtt cctggccaga ggaagacagg accagtacat caaagcctgc
     4741 agacactgcg atgctgacat cgaaagctgt gcacatatca tcggcaactg ccccgtgacc
     4801 caggacgccc gcataaagcg tcacaactat atctgcgagc ttctgctgga agaagccaaa
     4861 aagaaagatt gggtggtgtt taaggaacct cacatccggg acagcaacaa ggagctgtac
     4921 aagcctgatc tgatcttcgt caaggacgcc agagctctgg ttgtggacgt gactgtgaga
     4981 tacgaggccg ctaagtcctc cctggaagag gccgccgccg agaaggtgag aaagtacaaa
     5041 cacctggaaa ccgaggtgag acacctgacc aacgccaaag acgtgacctt cgtcggtttc
     5101 cctctgggcg ctcgcggcaa gtggcaccag gacaacttca agctcctgac tgaactggga
     5161 ctgtctaaaa gcagacaggt gaagatggca gagacatttt ccaccgtggc tctgttctcc
     5221 tccgtggaca tcgtccacat gttcgccagc agagcccgaa aaagcatggt gatggctagc
     5281 ccaaagaaga agaggaaagt cggatccgac tacaaagacc atgacggtga ttataaagat
     5341 catgacatcg attacaagga tgacgatgac aagtaataag ctgccttctg cggggcttgc
     5401 cttctggcca tgcccttctt ctctcccttg cacctgtacc tcttggtctt tgaataaagc
     5461 ctgagtagga agaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5521 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5581 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5641 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5701 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5761 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5821 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5881 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5941 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6001 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6061 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6121 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6181 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6241 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6301 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6361 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6421 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6481 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6541 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6601 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6661 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6721 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6781 aatgataccg cgtgagccac gctcaccggc tccagattta tcagcaataa accagccagc
     6841 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6901 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6961 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7021 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7081 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7141 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7201 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7261 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7321 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7381 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7441 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7501 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7561 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7621 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     7681 taaaaatagg cgtatcacga ggccctttcg tc
//
