LOCUS       Exported                6354 bp ds-DNA     circular SYN 19-AUG-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6354)
  AUTHORS   Atsuko Uchida
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Aug 21, 2019 from SnapGene Viewer 4.3.11
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6354
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV Forward
     primer_bind     559..577
                     /label=Sequencing F1
     misc_feature    653..658
                     /label=Kozak
     CDS             659..2284
                     /codon_start=1
                     /label=wt rat NFL
                     /translation="MSSFSYEPYFSTSYKRRYVETPRVHISSVRSGYSTARSAYSSYSA
                     PVSSSLSVRRSYSSSSGSLMPSLENLDLSQVAAISNDLKSIRTQEKAQLQDLNDRFASF
                     IERVHELEQQNKVLEAELLVLRQKHSEPSRFRALYEQEIRDLRLAAEDATNEKQALQGE
                     REGLEETLRNLQARYEEEVLSREDAEGRLMEARKGADEAALARAELEKRIDSLMDEIAF
                     LKKVHEEEIAELQAQIQYAQISVEMDVSSKPDLSAALKDIRAQYEKLAAKNMQNAEEWF
                     KSRFTVLTESAAKNTDAVRAAKDEVSESRRLLKAKTLEIEACRGMNEALEKQLQELEDK
                     QNADISAMQDTINKLENELRSTKSEMARYLKEYQDLLNVKMALDIEIAAYRKLLEGEET
                     RLSFTSVGSITSGYSQSSQVFGRSAYSGLQSSSYLMSARAFPAYYTSHVQEEQSEVEET
                     IEATKAEEAKDEPPSEGEAEEEEKEKEEGEEEEGAEEEEAAKDESEDAKEEEGGEGEEE
                     DTKESEEEEKKEESAGEEQAAKKKD"
     misc_feature    659..661
                     /label=start
     primer_bind     complement(663..696)
                     /label=rP8R mutagenesis Reverse
     primer_bind     663..696
                     /label=rP8R mutagenesis Forward
     primer_bind     complement(708..739)
                     /label=rP22S mutagenesis Reverse
     primer_bind     708..739
                     /label=rP22S mutagensis Forward
     primer_bind     complement(933..964)
                     /label=rN98S mutagenesis primer Reverse
     primer_bind     933..964
                     /label=rN98S mutagenesis primer Forward
     primer_bind     1071..1092
                     /label=Rat F1 484 primer
     primer_bind     complement(1448..1481)
                     /label=rL268P mutagenesis primer Reverse
     primer_bind     1448..1481
                     /label=rL268P mutagenesis primer Forward
     primer_bind     1464..1485
                     /label=Rat F2 877
     primer_bind     complement(1636..1675)
                     /label=rQ333P mutagenesis primer Reverse
     primer_bind     1636..1675
                     /label=rQ333P mutagenesis primer Forward
     primer_bind     complement(1830..1863)
                     /label=rE396K mutagenesis primer Reverse
     primer_bind     1830..1863
                     /label=rE396K mutagenesis primer Forward
     primer_bind     1941..1962
                     /label=Rat F3 1354 primer
     primer_bind     complement(1963..1997)
                     /label=rP440L mutagensis primer Reverse
     primer_bind     1963..1997
                     /label=rP440L mutagensis primer Forward
     N_region        2285..2299
                     /label=linker
     CDS             2300..3019
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    3142..3263
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3179..3198)
                     /label=SV40pA-R
     rep_origin      complement(3270..3725)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3377..3395)
                     /label=Sequencing R2
     promoter        3752..3856
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3858..4215
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4066..4201
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4250..5044
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    5276..5323
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5652..6240
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attcctgagg ggcgcaccga ccgccaccat
      661 gagttcgttc agctacgagc cgtacttttc gacctcctac aagcggcgct acgtggagac
      721 gccccgggtg cacatctcca gcgtgcgcag cggctacagc acggcgcgct ctgcgtactc
      781 cagctactcc gcgcccgtct cctcctctct gtcggtgcgc cgcagctact catccagctc
      841 cggctctttg atgcccagcc tggagaacct cgatctgagc caggtagccg ccatcagcaa
      901 cgacctcaag tctatccgca cacaggagaa ggcacagctg caggacctca acgatcgctt
      961 cgccagcttc atcgagcgcg tgcacgagct ggagcagcag aacaaggtcc tggaagccga
     1021 gctgttggtg ctgcgccaga agcactcaga gccttcccgc ttccgcgccc tgtatgagca
     1081 ggagatccgt gatctgcgac tggcggccga agacgccact aacgagaagc aggcgctgca
     1141 gggcgagcgc gaggggctgg aggagactct gcgcaacctg caggctcgct acgaggagga
     1201 ggtgctgagc cgcgaggacg ccgagggccg gctgatggaa gcccgcaaag gcgcggatga
     1261 ggctgcgctc gcccgcgccg agctggagaa gcgcatcgac agcctgatgg acgagatagc
     1321 cttcctgaaa aaggtgcacg aggaagagat cgccgagctg caggctcaga tccagtatgc
     1381 tcagatctcc gtggagatgg acgtgtcctc caagcccgac ctctccgccg ctctcaagga
     1441 catccgcgct cagtacgaga agctggccgc caagaatatg cagaatgccg aagagtggtt
     1501 caagagccgc ttcacggtgc taaccgagag cgccgccaag aacaccgacg cagtgcgcgc
     1561 tgccaaggac gaggtgtcgg aaagccgccg cctacttaag gctaagaccc tggagatcga
     1621 agcctgccgg ggtatgaacg aagctctaga gaagcagctg caggagctgg aggacaagca
     1681 gaatgcagac atcagcgcca tgcaggacac aatcaacaaa ctggagaatg agctgcgaag
     1741 cacgaagagc gagatggcca ggtacctgaa ggagtaccag gacctcctca atgtcaagat
     1801 ggcattggac attgagattg cagcttacag gaaactcttg gaaggcgaag aaaccaggct
     1861 cagtttcacc agcgtgggta gcataaccag cggctactct cagagctcgc aggtctttgg
     1921 ccgttctgct tacagtggct tgcagagcag ctcctacttg atgtctgctc gagcattccc
     1981 agcctactat accagccacg tccaggaaga gcagtcagag gtggaggaga ccattgaggc
     2041 tacgaaagct gaggaggcca aggatgagcc cccctctgaa ggagaagcag aagaggagga
     2101 gaaggagaaa gaggaggggg aggaagagga aggtgctgag gaggaagaag ctgccaagga
     2161 tgagtctgaa gatgccaaag aagaagaagg tggtgagggt gaagaggaag acaccaaaga
     2221 gtcagaagag gaagagaaga aagaggaaag tgctggggag gagcaagctg ctaagaagaa
     2281 agatggatcc atcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     2341 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     2401 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2461 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     2521 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2581 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2641 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2701 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2761 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2821 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2881 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     2941 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     3001 ggacgagctg tacaagtaaa gcggccgcga ctctagatca taatcagcca taccacattt
     3061 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     3121 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     3181 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3241 tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt
     3301 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     3361 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     3421 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     3481 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     3541 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     3601 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     3661 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     3721 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3781 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3841 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg
     3901 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3961 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4021 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4081 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4141 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4201 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4261 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4321 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4381 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
     4441 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4501 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4561 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4621 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4681 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4741 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
     4801 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4861 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4921 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4981 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     5041 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     5101 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     5161 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg
     5221 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca
     5281 ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg
     5341 tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc
     5401 gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc
     5461 aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat
     5521 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5581 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5641 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5701 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5761 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     5821 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5881 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5941 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6001 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6061 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6121 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6181 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6241 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6301 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcat
//
