LOCUS       sequence-200285-        7030 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 7030)
  AUTHORS   Cai D, Cohen KB, Luo T, Lichtman JW, Sanes JR
  TITLE     Improved tools for the Brainbow toolbox.
  JOURNAL   Nat Methods. 2013 May 5;10(6):540-7. doi: 10.1038/nmeth.2450. Epub 
            2013 May 5.
  PUBMED    23817127
REFERENCE   2  (bases 1 to 7030)
  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.2450"; journalName: "Nat Methods"; date: "2013-05-5- 
            5"; volume: "10"; issue: "6"; pages: "540-7"
COMMENT     Sequence Label: AAV-EF1a-BbChT
FEATURES             Location/Qualifiers
     source          1..7030
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   21..161
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   21..150
                     /label=AAV2 ITR
     rep_origin      236..691
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(323..342)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     533..554
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(708..727)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     827..849
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(887..905)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        973..1077
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1078..1146,1147..1938)
                     /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(1296..1315)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2109..2697
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2598..2617
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2759..2888
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2989..4167
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3220..4158
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4115..4135
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(4174..4207)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(4286..4993)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     4830..4848
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      4990..4999
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    5000..5033
                     /label=loxKR3
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(5037..5070)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(5149..5856)
                     /codon_start=1
                     /product="monomeric teal (cyan) fluorescent protein (Ai et 
                     al., 2006)"
                     /label=mTFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKKTTGWDASTERMYVRDGVLK
                     GDVKHKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYESA
                     VARNSTDGMDELYK"
     regulatory      5853..5862
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    5863..5896
                     /label=loxKR3
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5915..6503
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5968..5988)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6386..6397)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6535..7011
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(6734..6753)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
ORIGIN
        1 cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
       61 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
      121 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
      181 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
      241 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
      301 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
      361 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
      421 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
      481 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
      541 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
      601 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
      661 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
      721 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
      781 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
      841 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
      901 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
      961 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1021 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     1081 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1141 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1201 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1261 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1321 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1381 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     1441 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1501 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1561 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     1621 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     1681 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     1741 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     1801 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     1861 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     1921 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     1981 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     2041 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2101 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2161 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2221 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     2281 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2341 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2401 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2461 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2521 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2581 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     2641 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2701 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc
     2761 tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg
     2821 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     2881 gggttcctgc ggccgcacgc gtaagctttg caaagatgga taaagtttta aacagagagg
     2941 aatctttgca gctaatggac cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc
     3001 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     3061 aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     3121 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     3181 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc
     3241 cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg
     3301 ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg
     3361 ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg
     3421 ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
     3481 ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt
     3541 gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac
     3601 ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
     3661 agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg
     3721 ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg
     3781 gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
     3841 ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc
     3901 gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc
     3961 ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
     4021 actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa
     4081 tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt
     4141 ttttttcttc catttcaggt gtcgtgaggt acctaccgtt cgtatagcat acattatacg
     4201 aagttattta ggagagcaca cacttgcagc tcatgcagcc ggggccactc tcatcaggag
     4261 ggttcagctt actcacgcgt gcactcttgt acagctcgtc catgccgccg gtggagtggc
     4321 ggccctcggc gcgttcgtac tgttccacga tggtgtagtc ctcgttgtgg gaggtgatgt
     4381 ccaacttgat gttgacgttg taggcgccgg gcagctgcac gggcttcttg gccttgtagg
     4441 tggtcttgac ctcagcgtcg tagtggccgc cgtccttcag cttcagcctc tgcttgatct
     4501 cgcccttcag ggcgccgtcc tcggggtaca tccgctcgga ggaggcctcc cagcccatgg
     4561 tcttcttctg cattacgggg ccgtcggagg ggaagttggt gccgcgcagc ttcaccttgt
     4621 agatgaactc gccgtcctgc agggaggagt cctgggtcac ggtcaccacg ccgccgtcct
     4681 cgaagttcat cacgcgctcc cacttgaagc cctcggggaa ggacagcttc aagtagtcgg
     4741 ggatgtcggc ggggtgcttc acgtaggcct tggagccgta catgaactga ggggacagga
     4801 tgtcccaggc gaagggcagg gggccaccct tggtcacctt cagcttggcg gtctgggtgc
     4861 cctcgtaggg gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg ttcacggagc
     4921 cctccatgtg caccttgaag cgcatgaact ccttgatgat ggccatgtta tcctcctcgc
     4981 ccttgctcac catggtggca taacaaggta taatgtatgc tatacgaagt tatgatataa
     5041 cttcgtatag catacattat acgaacggta tcaggagagc acacacttgc agctcatgca
     5101 gccggggcca ctctcatcag gagggttcag cttactcacg cgtgcactct tgtacagctc
     5161 gtccatgccg tcggtggagt tgcgggccac ggcgctctcg taaacggtca ccttgttgta
     5221 gtccttgtcg tggttcagga tctcgatgcg gtggtccaca aagtgatagt cgggcagctt
     5281 caccgccttc ttggccctgt agatggtctt gaagtcaacg cggtggtggc cgccgccctc
     5341 cagcagcagc ttgtgcttga cgtcgccctt cagcacgccg tcgcgcacgt acatcctctc
     5401 ggtggaggcg tcccagccgg tggtcttctt ctgcatcacg gggccgttgg gggggaagtt
     5461 ctcgcccttg aggtgtatct cgtagatgaa ggagtcctcc tccatggaga tgtcggactt
     5521 caccttcacg atgcccttgt cctcgaaggt catggtgcgc tcccaagagt agccctcggg
     5581 gaaggactgc ttgaagtagt tggggatgtc gtcggggtac ttggtgaagg ccctgttgcc
     5641 gtaggcgaac gcggtggtca gaatgtcgta ggagaagggc aggggggctc cctccttcac
     5701 ctccaggttg atggtgttgg tgccgtcgta gggcttgccc tcgccctcgc cctcgatcac
     5761 gaaggcgtgg ccattcacgt tgccctccat cttcagcttg atcttcatgt cgggcttgat
     5821 tacgcccatt gtggtctcct cgcccttgct caccatggtg gcataacaag gtataatgta
     5881 tgctatacga agttatgata tcaagcttat cgataatcaa cctctggatt acaaaatttg
     5941 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     6001 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     6061 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     6121 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     6181 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     6241 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     6301 gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg
     6361 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     6421 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     6481 ctccctttgg gccgcctccc cgcatcgata ccgagcgctg ctcgagagat ctacgggtgg
     6541 catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc
     6601 accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat
     6661 aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt
     6721 agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact
     6781 gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga
     6841 ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc
     6901 accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct
     6961 cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg
     7021 taggtaacca
//
