LOCUS       sequence-358166-        6315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For making cell lines for titering pRV∆L-5tTA virus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6315)
  AUTHORS   Jin L, Sullivan HA, Zhu M, Lea NE, Lavin TK, Fu X, Matsuyama M, Hou 
            Y, Feng G, Wickersham IR
  TITLE     Third-generation rabies viral vectors allow nontoxic retrograde 
            targeting of projection neurons with greatly increased efficiency.
  JOURNAL   Cell Rep Methods. 2023 Nov 20;3(11):100644. doi: 
            10.1016/j.crmeth.2023.100644.
  PUBMED    37989085
REFERENCE   2  (bases 1 to 6315)
  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"; journalName: "Cell Rep 
            Methods."; date: "2023-11-20"; volume: "3(11):100644. doi"; pages: "
            10.1016/j.crmeth.2023.100644"
COMMENT     Sequence Label: pB-TREtight-EGFP
FEATURES             Location/Qualifiers
     source          1..6315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(27..131)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(157..612)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(294..315)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     506..525
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     739..761
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     753..770
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     754..770
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   900..962
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1386..1700
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1394..1412
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1430..1448
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1465..1483
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1501..1519
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1537..1555
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1572..1590
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1608..1626
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1673..1697
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1714..1723
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1720..1722,1723..1725,1726..2439)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1720..1722,1723..1725,1726..2436)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1765..1786)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2026..2045)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2373..2394
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2448..3036
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2501..2521)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2919..2930)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3040..3264
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3795..3829
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4320..4336)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4320..4336)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4333..4355)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4344..4360
                     /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)."
     promoter        complement(join(4368..4374,4375..4392,4393..4398))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4413..4434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4551..4568)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4722..5310)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4802..4821)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5481..6272,6273..6315,1..26))
                     /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     6104..6123
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
       61 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
      121 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
      181 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
      241 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
      301 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
      361 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
      421 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
      481 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
      541 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
      601 tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
      661 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
      721 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcct
      781 cgttcattca cgtttttgaa cccgtggagg acgggcagac tcgcggtgca aatgtgtttt
      841 acagcgtgat ggagcagatg aagatgctcg acacgctgca gaacacgcag ctagattaac
      901 cctagaaaga taatcatatt gtgacgtacg ttaaagataa tcatgtgtaa aattgacgca
      961 tgtgttttat cggtctgtat atcgaggttt atttattaat ttgaatagat attaagtttt
     1021 attatattta cacttacata ctaataataa attcaacaaa caatttattt atgtttattt
     1081 atttattaaa aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa cttttatgag
     1141 ggacagcccc cccccaaagc ccccagggat gtaattacgt ccctcccccg ctagggggca
     1201 gcagcgagcc gcccggggct ccgctccggt ccggcgctcc ccccgcatcc ccgagccggc
     1261 agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc ctctgaacgc
     1321 ttctcgctgc tctttgagcc tgcagacacc tggggggata cggggaaaag gcctccaacg
     1381 gtctcgagtt tactccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt
     1441 gatagagaac gatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac
     1501 tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta
     1561 tgtcgagttt atccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga
     1621 tagagaacgt atgtcgaggt aggcgtgtac ggtgggaggc ctatataagc agagctcgtt
     1681 tagtgaaccg tcagatcgcc tggagaattc gccgccacca tggtgagcaa gggcgaggag
     1741 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1801 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1861 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1921 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1981 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2041 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2101 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2161 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2221 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2281 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     2341 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2401 gccgggatca ctctcggcat ggacgagctg tacaagtaat aagatccaat caacctctgg
     2461 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     2521 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     2581 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     2641 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     2701 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     2761 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     2821 attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca
     2881 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     2941 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     3001 agacgagtcg gatctccctt tgggccgcct ccccgcctgc tgtgccttct agttgccagc
     3061 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3121 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3181 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3241 ctggggatgc ggtgggctct atggagatcc gcggcccatt ggtatggctt tttccccgta
     3301 tccccccagg tgtctgcagg ctcaaagagc agcgagaagc gttcagagga aagcgatccc
     3361 gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg atgcgggggg
     3421 agcgccggac cggagcggag ccccgggcgg ctcgctgctg ccccctagcg ggggagggac
     3481 gtaattacat ccctgggggc tttggggggg ggctgtccct gatatctata acaagaaaat
     3541 atatatataa taagttatca cgtaagtaga acatgaaata acaatataat tatcgtatga
     3601 gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca cgcggtcgtt
     3661 atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg agctccaagc
     3721 ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa agagagagca
     3781 atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt taatctagct
     3841 gcatcaggat catatcgtcg ggtctttttt ccggctcagt catcgcccaa gctggcgcta
     3901 tctgggcatc ggggaggaag aagcccgtgc cttttcccgc gaggttgaag cggcatggaa
     3961 agagtttgcc gaggatgact gctgctgcat tgacgttgag cgaaaacgca cgtttaccat
     4021 gatgattcgg gaaggtgtgg ccatgcacgc ctttaacggt gaactgttcg ttcaggccac
     4081 ctgggatacc agttcgtcgc ggcttttccg gacacagttc cggatggtca gcccgaagcg
     4141 catcagcaac ccgaacaata ccggcgacag ccggaactgc cgtgccggtg tgcagattaa
     4201 tgacagcggt gcggcgctgg gatattacgt cagcgaggac gggtatcctg gctggatgcc
     4261 gcagaaatgg acatggatac cccgtgagtt acccggcggg cgcgcttggc gtaatcatgg
     4321 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4381 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4441 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4501 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4561 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4621 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4681 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4741 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4801 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4861 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4921 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4981 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5041 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5101 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5161 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5221 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5281 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5341 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5401 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5461 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5521 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5581 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5641 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5701 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5761 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5821 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5881 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5941 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6001 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6061 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6121 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6181 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6241 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6301 aaaaagggaa taagg
//
