LOCUS       Exported                6562 bp ds-DNA     circular SYN 06-OCT-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6562)
  AUTHORS   Department of Anatomy
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, May 13, 2021 from SnapGene Viewer 5.2.5.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6562
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     promoter        1..448
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     promoter        468..486
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     CDS             521..538
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             542..574
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             578..601
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      608..617
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             629..685
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     CDS             740..1033
                     /codon_start=1
                     /label=TevCs
                     /translation="MSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVG
                     IHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFM"
     primer_bind     915..940
                     /label=TevC-M-FW
     primer_bind     complement(974..994)
                     /label=TevC-C-RV
     primer_bind     complement(1015..1036)
                     /label=CRE  vector RV
     primer_bind     1017..1053
                     /label=TevC-IRES FW
     misc_feature    1043..1595
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     1444..1461
                     /label=IRES FW
     primer_bind     complement(1583..1626)
                     /label=IRES-SP6 RV
     promoter        1602..1620
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1607..1651
                     /label=SP6-CRE FW
     regulatory      1621..1630
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1627..2658
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MANLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     primer_bind     complement(2634..2678)
                     /label=Cre-stop RV
     primer_bind     2659..2682
                     /label=CRE vector FW
     misc_feature    2685..3273
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2738..2758)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3156..3167)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3314..3521
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3691..3820
                     /label=AAV2 ITR
     primer_bind     complement(3737..3748)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3798..3809
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3886..3903)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4057..4645)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4137..4156)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4816..5676)
                     /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"
     primer_bind     5439..5458
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5677..5781)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5807..6262)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5944..5965)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6156..6175
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6334..6463
                     /label=AAV2 ITR
     primer_bind     complement(6345..6356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6406..6417
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga
       61 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga
      121 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg
      181 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc
      241 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg
      301 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg
      361 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg
      421 aggagtcgtg tcgtgcctga gagcgcagtc gagaggatct aagcttgatt taggtgacac
      481 tatagaatac aagctacttg ttctttttgc aggatccatg catcatcatc atcatcatgg
      541 tatggctagc atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa
      601 ggatctcgcc accatggtcg actcatcacg tcgtaagtgg aataagacag gtcacgcagt
      661 cagagctata ggtcggctga gctcactcga aggtggcggt ggctctggag gtggtgggtc
      721 cggaggaggc ggcatcgaga tgtccagcat ggtgagcgat actagctgta ccttcccatc
      781 atctgacgga atcttctgga agcactggat tcagactaag gacggccagt gtggcagccc
      841 actggtgagc acacgagacg gattcatcgt ggggattcac agcgcctcca actttacaaa
      901 caccaataac tatttcacct cagtgccaaa gaactttatg gagctgctga ccaaccagga
      961 ggcccagcag tgggtgagcg ggtggcgcct gaacgccgat tccgtgctgt ggggcgggca
     1021 caaggtgttt atgtagctcg agacgttact ggccgaagcc gcttggaata aggccggtgt
     1081 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
     1141 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
     1201 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa
     1261 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
     1321 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
     1381 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
     1441 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
     1501 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg
     1561 gacgtggttt tcctttgaaa aacacgatga taatagaatt catttaggtg acactataga
     1621 gccaccatgg ccaatttact gaccgtacac caaaatttgc ctgcattacc ggtcgatgca
     1681 acgagtgatg aggttcgcaa gaacctgatg gacatgttca gggatcgcca ggcgttttct
     1741 gagcatacct ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg gtgcaagttg
     1801 aataaccgga aatggtttcc cgcagaacct gaagatgttc gcgattatct tctatatctt
     1861 caggcgcgcg gtctggcagt aaaaactatc cagcaacatt tgggccagct aaacatgctt
     1921 catcgtcggt ccgggctgcc acgaccaagt gacagcaatg ctgtttcact ggttatgcgg
     1981 cggatccgaa aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct agcgttcgaa
     2041 cgcactgatt tcgaccaggt tcgttcactc atggaaaata gcgatcgctg ccaggatata
     2101 cgtaatctgg catttctggg gattgcttat aacaccctgt tacgtatagc cgaaattgcc
     2161 aggatcaggg ttaaagatat ctcacgtact gacggtggga gaatgttaat ccatattggc
     2221 agaacgaaaa cgctggttag caccgcaggt gtagagaagg cacttagcct gggggtaact
     2281 aaactggtcg agcgatggat ttccgtctct ggtgtagctg atgatccgaa taactacctg
     2341 ttttgccggg tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca gctatcaact
     2401 cgcgccctgg aagggatttt tgaagcaact catcgattga tttacggcgc taaggatgac
     2461 tctggtcaga gatacctggc ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat
     2521 atggcccgcg ctggagtttc aataccggag atcatgcaag ctggtggctg gaccaatgta
     2581 aatattgtca tgaactatat ccgtaacctg gatagtgaaa caggggcaat ggtgcgcctg
     2641 ctggaagatg gcgattaatc tagatagcta gccagcttat cgataatcaa cctctggatt
     2701 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     2761 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     2821 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     2881 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     2941 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     3001 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3061 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     3121 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3181 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     3241 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgaccc gggcggccgc
     3301 tcataaagct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3361 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3421 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3481 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtccg gactgtacaa
     3541 ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta
     3601 aaacctctac aaatgtggta aaatcgataa ggatcttcct agagcatggc tacgtagata
     3661 agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc
     3721 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     3781 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcattaat gaatcggcca
     3841 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3901 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3961 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4021 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4081 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4141 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4201 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4261 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4321 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4381 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4441 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4501 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4561 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4621 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4681 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4741 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4801 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4861 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4921 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4981 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5041 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5101 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5161 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5221 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5281 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5341 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5401 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5461 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5521 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5581 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5641 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5701 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5761 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     5821 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     5881 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     5941 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     6001 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     6061 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     6121 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     6181 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     6241 aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg
     6301 cgatcggtgc gggcctcttc gctattacgc cagctgcgcg ctcgctcgct cactgaggcc
     6361 gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
     6421 gcgcgcagag agggagtggc caactccatc actaggggtt ccttgtagtt aatgattaac
     6481 ccgccatgct acttatctac gtagccatgc tctaggaaga tcgtaggtta attaatctag
     6541 actgcagagg gccctgcgta tg
//
