LOCUS       sequence-116742-        7187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7187)
  AUTHORS   Kim J, Zhao T, Petralia RS, Yu Y, Peng H, Myers E, Magee JC
  TITLE     mGRASP enables mapping mammalian synaptic connectivity with light 
            microscopy.
  JOURNAL   Nat Methods. 2011 Dec 4;9(1):96-102. doi: 10.1038/nmeth.1784.
  PUBMED    22138823
REFERENCE   2  (bases 1 to 7187)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nmeth"; 
            journalName: "Nat Methods"; date: "2011-12-4- 4"; volume: "9"; 
            issue: "1"; pages: "96-102"
COMMENT     Sequence Label: paavCAG-pre-mGRASP-mCerulean-2A-nls-mCherry
FEATURES             Location/Qualifiers
     source          1..7187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    273..394
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(310..329)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     364..383
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    437..1025
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             543..554
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     952..972
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1036..1743)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1116..1139)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1314..1333)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1583..1601
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1744..1764)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1786..1839)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(3424..3471)
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     primer_bind     complement(3597..3616)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(3678..3700)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(3979..4235)
                     /label=chicken beta-actin promoter
                     /note="contains a 21-bp deletion relative to the standard 
                     chicken beta-actin promoter"
     repeat_region   4548..4677
                     /label=AAV2 ITR
     rep_origin      4749..5204
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4836..4855)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5046..5067
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5230..5334
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5335..5403,5404..6195)
                     /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     complement(5553..5572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6366..6954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6855..6874
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg gcctcagtga
       61 gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc ttgtagttaa
      121 tgattaaccc gccatgctac ttatctacgt agccatgctc taggaagatc cttatcgatt
      181 ttaccacatt tgtagaggtt ttacttgctt taaaaaacct cccacatctc cccctgaacc
      241 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
      301 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
      361 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgctcgaag cggccgcccg
      421 ggtcgacggt atcgatgcgg ggaggcggcc caaagggaga tccgactcgt ctgagggcga
      481 aggcgaagac gcggaagagg ccgcagagcc ggcagcaggc cgcgggaagg aaggtccgct
      541 ggattgaggg ccgaagggac gtagcagaag gacgtcccgc gcagaatcca ggtggcaaca
      601 caggcgagca gccaaggaaa ggacgatgat ttccccgaca acaccacgga attgtcagtg
      661 cccaacagcc gagcccctgt ccagcagcgg gcaaggcagg cggcgatgag ttccgccgtg
      721 gcaataggga gggggaaagc gaaagtcccg gaaaggagct gacaggtggt ggcaatgccc
      781 caaccagtgg gggttgcgtc agcaaacaca gtgcacacca cgccacgttg cctgacaacg
      841 ggccacaact cctcataaag agacagcaac caggatttat acaaggagga gaaaatgaaa
      901 gccatacggg aagcaatagc atgatacaaa ggcattaaag cagcgtatcc acatagcgta
      961 aaaggagcaa catagttaag aataccagtc aatctttcac aaattttgta atccagaggt
     1021 tgattatcga taagcttact tgtacagctc gtccatgccg ccggtggagt ggcggccctc
     1081 ggcgcgctcg tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccagctt
     1141 gatgttgacg ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt
     1201 gacctcggcg tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt
     1261 cagggcgccg tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt
     1321 ctgcattacg gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa
     1381 ctcgccgtcc tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt
     1441 catcacgcgc tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc
     1501 ggcggggtgc ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca
     1561 ggcgaagggc agggggccgc ccttggtcac cttcagcttg gcggtctggg tgccctcgta
     1621 ggggcggccc tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat
     1681 gtgcaccttg aagcgcatga actccttgat gatggccatg ttgtcctcct cgcccttgct
     1741 cacgactttc ctcttcttct tgggcaccat ggtcgacccc ggggcgggac caggattctc
     1801 ttctacgtcg ccgcatgtca gcagactacc ccgtccttcg aattccttgt acagctcatc
     1861 catacccagg gttatgccgg cagctgtgac aaactccagc agtaccatat gatctctctt
     1921 ttcgttgggg tccttgctca gcttgctctg tgtactaagg tagtgattgt cgggcaacag
     1981 cacaggtcca tcaccgatgg gagtattctg ttggtaatgg tcagccagct gaacgcttcc
     2041 atcctctatg ttgtgccgga ttttgaaatt ggcctttatg ccatttttct gcttgtcggc
     2101 ggttatgtaa acattatcag atatggcgtt gtattccagc ttgtggccga gaatgttccc
     2161 atcttctttg aagtcgattc ccttgagttc aatcctgttc accaaggtgt caccttcgaa
     2221 tttcacctcc gcccttgtct tgtagttacc atcgtccttg aaaaatatcg tcctttcctg
     2281 aacatacccc tcgggcatag cggacttgaa gaagtcgtgc tgcttcatgt ggtcagggta
     2341 ccgggcgaag cactgtacac cccatgtgag ggtggtgacg agtgtgggcc aaggcacggg
     2401 gagcttccct gttgtacaga tgaacttcag cgtcagtttg ccgtaggtgg catccccttc
     2461 tccctcgccg cttacggaga acttgtgccc atttacatct ccgtcgagct ccaccaggat
     2521 aggcacgacc cctgtaaaca actcttcgcc cttgctcacc atagatctta cgtagtactc
     2581 tttatccttg tttttcttgt ttttgtttgc tgatttggcg ctggagggct gtttctcttt
     2641 aaccacggcg ccattactct gagcgctgtt actgatatag ttcctagatt cgtcaacgtg
     2701 gtaagatccc tcatccctgt tgcgatactt gtagcgtacg cagaagaaga tgcccaggcc
     2761 gataaacaga agcaggccgg cgactccccc gagtacgatc agggccatcg gctgtactgg
     2821 tgttgaccaa gtaggaagca ccttgatgtt gctctccagc agcacctgtc cagagtcgct
     2881 aagaagacac tgccacatcc ctgcctccgg gttaaggacc cacacagcct tctctctttt
     2941 actgacttta gcctccttat tctccagctt cagagacagc atcagtttag gtgaggtggg
     3001 accccacact tcgcaggtca ggttcttctg cagctgtgtt gccctcatca ccacgaggtt
     3061 gacctcctga tgcaactttc cggtcttagc ctccaaggcg agggtgaggt tcccggagcc
     3121 agcatactgg ggcagggctt gggggagtgt caagtgcaaa ggcaacttct tgcccatctg
     3181 cagcttaggg tcctgtgtga ctctcttcac gctcacttct ttgtttttca gatcaaaagt
     3241 gatccaactc ttgctggagg aggctctttc ggcctgccac cacaactctc ctgatcctgt
     3301 gagcttctcg actgtgaagg ccaatgggaa actgaactcc acctgttccc cctccttctt
     3361 ataaacaata gagctagctt tctgaaagtc gacgctacca ccgccgccag atccgccacc
     3421 gcctgtgatg ccagcagcgt tcacatattc gtgcagcacc atgtggtcgc gggaagcggt
     3481 gacttctcct gtcttccaat ctgaagcggg aagagcgaag gggagaagtg cagcaagcaa
     3541 caaaagacta gtagaaggtg gcatggatcc aattctttgc caaaatgatg agacagcaca
     3601 ataaccagca cgttgcccag gagctgtagg aaaaagaaga aggcatgaac atggttagca
     3661 gaggctctag agccgccggt cacacgccag aagccgaacc ccgccctgcc ccgtcccccc
     3721 cgaaggcagc cgtccccccg cggacagccc cgagccgctc cccccgcaca aagggccctc
     3781 ccggagcccc tcaaggcttt cacgcagcca cagaaaagaa acaagccgtc attaaaccaa
     3841 gcgctaatta cagcccggag gagaagggcc gtcccgcccg ctcacctgtg ggagtaacgc
     3901 ggtcagtcag agccggggcg ggcggcgcga ggcggcggcg gagcggggca cggggcgaag
     3961 gcagcgcgca gcgactcccg cccgccgcgc gcttcgcttt ttatagggcc gccgccgccg
     4021 ccgcctcgcc ataaaaggaa actttcggag cgcgccgctc tgattggctg ccgccgcacc
     4081 tctccgcctc gccccgcccc gcccctcgcc ccgccccccc cccccccccg cccccatcgc
     4141 tgcacaaaat aattaaaaaa taaataaata caaaattggg ggtggggagg ggggggagat
     4201 ggggagagtg aagcagaacg tggggctcac ctcgaccatg gtaatagcga tgactaatac
     4261 gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc
     4321 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     4381 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc
     4441 tattggcgtt actatgggaa catacgtcat tattgacgtc aatggtacga tcttcctaga
     4501 gcatggctac gtagataagt agcatggcgg gttaatcatt aactacaagg aacccctagt
     4561 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     4621 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     4681 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     4741 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4801 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4861 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4921 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4981 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5041 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5101 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5161 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     5221 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5281 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5341 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5401 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5461 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5521 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5581 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5641 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5701 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5761 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5821 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5881 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5941 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6001 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6061 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6121 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6181 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6241 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6301 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6361 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6421 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6481 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6541 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6601 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6661 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6721 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6781 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6841 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6901 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6961 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7021 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7081 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     7141 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatg
//
