LOCUS       sequence-12314-e        7403 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 7403)
  AUTHORS   Wall NR, Wickersham IR, Cetin A, De La Parra M, Callaway EM
  TITLE     Monosynaptic circuit tracing in vivo through Cre-dependent targeting
            and complementation of modified rabies virus.
  JOURNAL   Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21848-53. doi: 
            10.1073/pnas.1011756107
  PUBMED    21115815
REFERENCE   2  (bases 1 to 7403)
  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: "10.1073/pnas"; 
            journalName: "Proc Natl Acad Sci U S A"; date: "2010-12-14- 14"; 
            volume: "107"; issue: "50"; pages: "21848-53"
COMMENT     Sequence Label: pAAV-FLEX-hGTB
FEATURES             Location/Qualifiers
     source          1..7403
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     278..300
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     292..309
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     293..309
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     320..339
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        320..338
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             343..354
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    join(584..602,603..625,626..644,645..667,668..686,687..709,
                     710..728,729..751,752..770,771..793,794..812,813..835,
                     836..854)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     primer_bind     887..911
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      992..1001
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1004..1037
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1100..1133
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2724..2783)
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     CDS             complement(3273..3326)
                     /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(join(3342..4052,4053..4055))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(3342..4052,4053..4055))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3384..3405)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3733..3752
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3992..4013
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(4281..4898)
                     /codon_start=1
                     /gene="synthetic gene"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     protein_bind    complement(4910..4943)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(5014..5047)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    5080..5201
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5091..5110)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5145..5164
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5309..5438
                     /label=AAV2 ITR
     rep_origin      complement(5719..6307)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5799..5818)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6481..7275)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(7276..7380)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt
      181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca
      361 aggcctaggc gcgccctcga gttaattaat tggccactcc ctctctgcgc gctcgctcgc
      421 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
      481 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctggaggg
      541 gtaacgttgg ctcgacccgg gtaccgagct cgactttcac ttttctctat cactgatagg
      601 gagtggtaaa ctcgactttc acttttctct atcactgata gggagtggta aactcgactt
      661 tcacttttct ctatcactga tagggagtgg taaactcgac tttcactttt ctctatcact
      721 gatagggagt ggtaaactcg actttcactt ttctctatca ctgataggga gtggtaaact
      781 cgactttcac ttttctctat cactgatagg gagtggtaaa ctcgactttc acttttctct
      841 atcactgata gggagtggta aaggatccct cgacctatat aagcagagct cgtttagtga
      901 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg
      961 accgatccag cctccgcggc cctgcagggc cgccaccatg gccataactt cgtataaagt
     1021 atcctatacg aagttatatc aaaataggaa gaccaatgct tcaccatcga cccgaattgc
     1081 caagcatcac catcgaccca taacttcgta taatgtatgc tatacgaagt tatacttcaa
     1141 ctagttcaca gtctggtctc gccgccgctc ttgtggctct cccagctgct gatgatcttg
     1201 ccgctctggg gggtcacgct cacctctctg ccggtgcctc tcaggttgtg ctgggtgggc
     1261 tcgcttctgt tcactcttct gcagcaggtc atcaggaaga tgatcagcat cagggcggtc
     1321 agggcgccgg cgctcagcag cacgtacttg ccccagttgg gcaggcccag gtccacgccg
     1381 ctcacctggt tgtgcacgtc gggcaggtgc acctccacga agtcctcggc ctcgtcgccg
     1441 tccttgaaca cggtgctggg gtcggccagg gggtgcacca gggggatcac gctgctctcc
     1501 agcagctcca tgtgctgctg cagcaggctg ctctgcatct cggggatcag cacgttgccg
     1561 tcggggccca ggatgatgcc gttgaagaac acgccgttca cgtgggggtg gcatctgccg
     1621 cccactctca ggcagccctt gctgggcagg atctcgttcc aggttctcac gctcttgtag
     1681 tgggcgtcgg cctccatcag ggtcttgttg aagatggtgt aggccttgcc gaagccgggc
     1741 accagctttc tcaggtggct cagtcttctg aagctcacgc tcttggtggt catgatgctc
     1801 tccagggcgt ccaggcactc ctctctcttt ctcaccagct cctccaccac caggtgctcg
     1861 atctcgtcgc ttctgaagtc gtgcaggttc accagcttgt cgggggggca ccacttggtc
     1921 tcgttgctgg tctgcatgct cacccaggtg ccgtccatca gtctcaggcc cagcacgccg
     1981 cacagcttca gcttgcaggc gcccttcagg ctcttgtaca ggcctctctc gtccacgaag
     2041 ccgcaggtct cgctgccctt gctggctctc ttgcctctgc tgttggtgaa gatgtcgcag
     2101 ctcatgccca gtctggggtt ctcgggcatc cagatggtgt agtcgtggtt ggtgctgcag
     2161 taggtgctgc tcacggccac gccgctgcac ttgccgctgg ggaacactct gctgtgcagg
     2221 cttctgtcgt aggggtccag gtcggccacg ctggggctga tgatcaccag gctctccttg
     2281 gtggtcttca cggttctcag ccatctgtag tcggggtagg ggttgtgcag gctctcctcg
     2341 tatctggggt cgccggccat cttccagttg taggcggctc tgcaggcgtc gggggtgggt
     2401 ctgaagtgct ttctcttgaa ggtggtggtc acgtagccca cgaagttggt gtaggtctcg
     2461 gcctcggtca ccacgccggt gcaggtgaag ccgttcacct tgatggccag gatgtagccc
     2521 accttcagct ccatgtagct gaagccgctc aggttggtgc agccctcgtc ctccaccacc
     2581 aggttgttgg ggcagctcag gtggtggatg tcgatggggc tccaggggcc cagcttgtcg
     2641 gggatggtgt agatggggaa cttgccgaag cacaggggga acaccagcag gggcacgaac
     2701 agcagggcct ggggcactcc ggaaggaccg gggttacttt caacatcgcc agcgagtttc
     2761 aacaaagcgt agttagtaca ttgaccacta ccgctagcgg agaacaagtc tgcctggctc
     2821 ttgtcaggca ccagcagctc cttggatgcg ctttcaagac tgaagatgtc agacctgctt
     2881 tttgctttgg acttccccca tgcagcgata ccacccacag ctaccaggca gcacaggagc
     2941 actgcagtga tcagcatcca catgcggccg tgattcctgg ctggcagagc acgtccagga
     3001 gcaggggcag tgggagcctc tgtgccgttg tccgtgggca ccgcggggat cgcgctggtc
     3061 ccgcagcccc actcgtcccg cccgtcgtcg cagtcggggt gtccgtcgca cagccagtcc
     3121 tgcgggtaac actccccgtg ggcaccgggc ggctccgagc agcggaactg accggggggg
     3181 caacgggaca aagaaccgtt accggacccg ttaccggtca cgttaccggg cagcagcagc
     3241 agcagcagcg cgggcagcag ccgcgcatcg attgggccag gattctcctc gacgtcaccg
     3301 catgttagca gacttcctct gccctctcca ctaccaagct tcttgtacag ctcgtccatg
     3361 ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg
     3421 ttggggtctt tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg cagcagcacg
     3481 gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc
     3541 tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt tcttctgctt gtcggccatg
     3601 atatagacgt tgtggctgtt gtagttgtac tccagcttgt gccccaggat gttgccgtcc
     3661 tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc
     3721 acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg ctcctggacg
     3781 tagccttcgg gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg
     3841 ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg tgggccaggg cacgggcagc
     3901 ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc gccctcgccc
     3961 tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac caggatgggc
     4021 accaccccgg tgaacagctc ctcgcccttg ctcacctcga gtgggttgga ctccacgtct
     4081 cccgccaact tgagaaggtc aaaattcaaa gtctgtttca caccactacc acgcgtcagc
     4141 atatccaggt caaaatcatc aagtgcatca gcagggagca tgtccaaatc aaagtcatcc
     4201 agggcatcag caggcagcat gtccaggtca aaatcatcca aggcatcagc aggcacgtgc
     4261 tttctctttt tgggcacgtg agagccagac tcacatttca gctgcttttc caggccacag
     4321 atgatcagtt ccaggccaaa aaggaatgca ggctcagccc cttggtggtc aaacaactca
     4381 atagcttgcc gcagcagtgg tggcatggag tcagtggtgg gggtctccct ctcctccttg
     4441 gcaacttggt gctcctggtc ctccagcaca cagcccaagg tgaagtggcc cacagcagag
     4501 agggcataca aggcattttc cagggagaag ccttgttggc acaggaatgc cagctggttc
     4561 tccagggtct catactgctt ctctgtaggc ctggtgccca agtgaacttt ggcaccatcc
     4621 cggtgggaga gcaaggcaca tctgaagctc ttggcattgt tcctcaggaa gtcctgccag
     4681 gactcccctt ccagagggca gaagtgggtg tggtgcctgt ccaacatctc aatggccagg
     4741 gcatcaagca gagccctctt gttcttcaca tgccagtaca gggtaggctg ctccacaccc
     4801 agcttctggg ccagcttcct ggtggtcaag ccctcaatgc caacttcatt caggagctcc
     4861 aaggcagagt tgatgacttt gctcttgtcc agtctggaag tgtcgacgca taacttcgta
     4921 taggatactt tatacgaagt tatctttggg attcttccta ttttgatcca agcatcacca
     4981 tcgaccctct agtccagatc tcaccatcga cccataactt cgtatagcat acattatacg
     5041 aagttatgtc cctcgaagag gttcgaattc cagacatgat aagatacatt gatgagtttg
     5101 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     5161 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     5221 attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct
     5281 acaaatgtgg tatctagaat taactacaag gaacccctag tgatggagtt ggccactccc
     5341 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     5401 tttgcccggg cggcctcagt gagcgagcga gcgcgcagag agggagtggc caagcggccg
     5461 cttggatccc agaattctag gtacctctta attaactggc ctcatgggcc ttcctttcac
     5521 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaacatggt catagctgtt
     5581 tccttgcgta ttgggcgctc tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5641 gggtaaagcc tggggtgcct aatgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5701 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5761 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5821 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5881 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5941 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6001 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6061 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6121 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6181 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6241 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6301 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6361 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6421 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6481 ttagaaaaat tcatccagca gacgataaaa cgcaatacgc tggctatccg gtgccgcaat
     6541 gccatacagc accagaaaac gatccgccca ttcgccgccc agttcttccg caatatcacg
     6601 ggtggccagc gcaatatcct gataacgatc cgccacgccc agacggccgc aatcaataaa
     6661 gccgctaaaa cggccatttt ccaccataat gttcggcagg cacgcatcac catgggtcac
     6721 caccagatct tcgccatccg gcatgctcgc tttcagacgc gcaaacagct ctgccggtgc
     6781 caggccctga tgttcttcat ccagatcatc ctgatccacc aggcccgctt ccatacgggt
     6841 acgcgcacgt tcaatacgat gtttcgcctg atgatcaaac ggacaggtcg ccgggtccag
     6901 ggtatgcaga cgacgcatgg catccgccat aatgctcact ttttctgccg gcgccagatg
     6961 gctagacagc agatcctgac ccggcacttc gcccagcagc agccaatcac ggcccgcttc
     7021 ggtcaccaca tccagcaccg ccgcacacgg aacaccggtg gtggccagcc agctcagacg
     7081 cgccgcttca tcctgcagct cgttcagcgc accgctcaga tcggttttca caaacagcac
     7141 cggacgaccc tgcgcgctca gacgaaacac cgccgcatca gagcagccaa tggtctgctg
     7201 cgcccaatca tagccaaaca gacgttccac ccacgctgcc gggctacccg catgcaggcc
     7261 atcctgttca atcatactct tcctttttca atattattga agcatttatc agggttattg
     7321 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7381 cacatttccc cgaaaagtgc cac
//
