LOCUS       sequence-79876-e        6786 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pre-2Ato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6786)
  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 6786)
  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-2A-dTomato
FEATURES             Location/Qualifiers
     source          1..6786
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   259..388
                     /label=AAV2 ITR
     promoter        701..978
                     /label=chicken beta-actin promoter
     primer_bind     1258..1280
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1342..1361
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1487..1534
                     /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"
     CDS             2396..2449
                     /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"
     misc_feature    3180..3768
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3233..3253)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3651..3662)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3811..3932
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3822..3841)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3876..3895
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4094..4223
                     /label=AAV2 ITR
     primer_bind     complement(4242..4259)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4242..4258)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4251..4273)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4400..4855
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4487..4506)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4697..4718
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4881..4985
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4986..5054,5055..5846)
                     /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(5204..5223)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6017..6605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6506..6525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatcgta ccattgacgt caataatgac gtatgttccc
      481 atagtaacgc caatagggac tttccattga cgtcaatggg tggactattt acggtaaact
      541 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      601 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      661 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
      721 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
      781 taattatttt gtgcagcgat gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg
      841 gggcggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc
      901 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
      961 cgaagcgcgc ggcgggcggg agtcgctgcg acgctgcctt cgccccgtgc cccgctccgc
     1021 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg
     1081 gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc
     1141 ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag
     1201 cggctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt
     1261 tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct
     1321 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca
     1381 aagaattgga tccatgccac cttctactag tcttttgttg cttgctgcac ttctcccctt
     1441 cgctcttccc gcttcagatt ggaagacagg agaagtcacc gcttcccgcg accacatggt
     1501 gctgcacgaa tatgtgaacg ctgctggcat cacaggcggt ggcggatctg gcggcggtgg
     1561 tagcgtcgac tttcagaaag ctagctctat tgtttataag aaggaggggg aacaggtgga
     1621 gttcagtttc ccattggcct tcacagtcga gaagctcaca ggatcaggag agttgtggtg
     1681 gcaggccgaa agagcctcct ccagcaagag ttggatcact tttgatctga aaaacaaaga
     1741 agtgagcgtg aagagagtca cacaggaccc taagctgcag atgggcaaga agttgccttt
     1801 gcacttgaca ctcccccaag ccctgcccca gtatgctggc tccgggaacc tcaccctcgc
     1861 cttggaggct aagaccggaa agttgcatca ggaggtcaac ctcgtggtga tgagggcaac
     1921 acagctgcag aagaacctga cctgcgaagt gtggggtccc acctcaccta aactgatgct
     1981 gtctctgaag ctggagaata aggaggctaa agtcagtaaa agagagaagg ctgtgtgggt
     2041 ccttaacccg gaggcaggga tgtggcagtg tcttcttagc gactctggac aggtgctgct
     2101 ggagagcaac atcaaggtgc ttcctacttg gtcaacacca gtacagccga tggccctgat
     2161 cgtactcggg ggagtcgccg gcctgcttct gtttatcggc ctgggcatct tcttctgcgt
     2221 acgctacaag tatcgcaaca gggatgaggg atcttaccac gttgacgaat ctaggaacta
     2281 tatcagtaac agcgctcaga gtaatggcgc cgtggttaaa gagaaacagc cctccagcgc
     2341 caaatcagca aacaaaaaca agaaaaacaa ggataaagag tactacgtaa gatctgaggg
     2401 tcggggctct ctgctcacat gtggcgacgt cgaggagaat cccggaccgg ccccggggtc
     2461 gacaatggtg tctaagggtg aggaagtaat taaggagttt atgagattca aagttagaat
     2521 ggagggtagc atgaacgggc acgagtttga aattgaaggt gagggtgaag gaagaccata
     2581 tgagggcaca caaaccgcca agctcaaggt taccaaaggt ggcccccttc ctttcgcctg
     2641 ggacatcctc agccctcaat tcatgtacgg tagcaaggct tacgtcaaac accctgctga
     2701 tatacctgat tacaaaaaac tgtcatttcc tgaaggattt aagtgggaac gagtaatgaa
     2761 ttttgaggat ggtgggctgg tcactgtcac gcaagactcc tctctgcagg acggcaccct
     2821 gatttacaag gtgaagatgc gcggaacaaa ttttcccccc gatggccccg tgatgcagaa
     2881 gaagaccatg gggtgggagg cctctacaga acgactgtac ccccgcgatg gcgttcttaa
     2941 aggcgaaatc catcaggcgc ttaaactcaa agatggcggg cactacctgg ttgagttcaa
     3001 aactatatat atggccaaaa agccggttca gctgcctggc tactactacg tggacaccaa
     3061 gttggacatc acgagtcaca acgaggacta cacaatcgtc gagcagtacg aacgctcaga
     3121 aggtcggcat cacctgttct tgtatggcat ggatgagctg tataagtaag cttatcgata
     3181 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     3241 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     3301 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     3361 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     3421 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     3481 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     3541 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     3601 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     3661 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     3721 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc
     3781 gacccgggcg gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca
     3841 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     3901 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     3961 ttcaggttca gggggagatg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     4021 gtaaaatcga taaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta
     4081 atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4141 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4201 tcagtgagcg agcgagcgcg cagccttaat taacctaatt cactggccgt cgttttacaa
     4261 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     4321 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     4381 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4441 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     4501 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4561 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     4621 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4681 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     4741 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     4801 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     4861 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4921 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4981 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5041 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5101 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5161 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5221 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5281 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5341 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5401 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5461 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5521 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5581 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5641 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     5701 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     5761 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5821 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5881 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5941 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6001 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6061 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6601 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     6661 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     6721 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     6781 cggaag
//
