LOCUS       sequence-325372-        6523 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses TetON3G under hEF1alpha in TUPV3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6523)
  TITLE     Leonard Lab mMoClo Plasmids
REFERENCE   2  (bases 1 to 6523)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pJM422 EF1alpha TetON3G in TU3
FEATURES             Location/Qualifiers
     source          1..6523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        350..1532
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          581..1523
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1480..1500
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1534..1558
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1639..2385
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(2430..2447)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2436..2660
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3429..3857
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3516..3535)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3726..3747
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3880..4076
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3927..4062
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3989..4008
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4196..4317
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4233..4252)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4287..4306
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4366..4382)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4366..4382)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4379..4401)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4390..4406
                     /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(4414..4420,4421..4438,4439..4444))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4459..4480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4597..4614)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4768..5356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4848..4867)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5527..6318,6319..6387))
                     /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     6150..6169
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6388..6492)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatcgaag acggactaaa tagtagttca gacgccgtta agcgcaagat
       61 ctggggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
      121 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
      181 gggccagata tacgcgtgag taattcatac aaaaggactc gcccctgcct tggggaatcc
      241 cagggaccgt cgttaaactc ccactaacgt agaacccaga gatcgctgcg ttcccgcccc
      301 ctcacccgcc cgctctcgtc atcactgagg tggagaagag catgcgtgag gctccggtgc
      361 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
      421 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
      481 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
      541 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc
      601 ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacgcc
      661 cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt
      721 cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcttgggc
      781 gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata
      841 agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata
      901 gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg
      961 gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc
     1021 caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg
     1081 cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt
     1141 gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc
     1201 gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag
     1261 ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct
     1321 cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc
     1381 cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct
     1441 tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc
     1501 aaagtttttt tcttccattt caggtgtcgt gagagctcgt ttagtgaacc gtcagatcgc
     1561 ctggagacgc catccacgct gttttgacct ccatagaaga gaccgggacc gatccaaatt
     1621 ctcgaggcta gcgccaccat gtctagactg gacaagagca aagtcataaa ctctgctctg
     1681 gaattactca atggagtcgg tatcgaaggc ctgacgacaa ggaaactcgc tcaaaagctg
     1741 ggagttgagc agcctaccct gtactggcac gtgaagaaca agcgggccct gctcgatgcc
     1801 ctgccaatcg agatgctgga caggcatcat acccactcct gccccctgga aggcgagtca
     1861 tggcaagact ttctgcggaa caacgccaag tcataccgct gtgctctcct ctcacatcgc
     1921 gacggggcta aagtgcatct cggcacccgc ccaacagaga aacagtacga aaccctggaa
     1981 aatcagctcg cgttcctgtg tcagcaaggc ttctccctgg agaacgcact gtacgctctg
     2041 tccgccgtgg gccactttac actgggctgc gtattggagg aacaggagca tcaagtagca
     2101 aaagaggaaa gagagacacc taccaccgat tctatgcccc cacttctgaa acaagcaatt
     2161 gagctgttcg accggcaggg agccgaacct gccttccttt tcggcctgga actaatcata
     2221 tgtggcctgg agaaacagct aaagtgcgaa agcggcgggc cgaccgacgc ccttgacgat
     2281 tttgacttag acatgctccc agccgatgcc cttgacgact ttgaccttga tatgctgcct
     2341 gctgacgctc ttgacgattt tgaccttgac atgctccccg ggtaagcggc cgctcgagtc
     2401 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2461 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2521 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2581 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gtcaatagca ggcatgctgg
     2641 ggatgcggtg ggctctatgg cttctgaggc aggtcgaaga gactgcccga cacatcttag
     2701 tgagcgcatc gcgataagct ctagagggac agcccccccc caaagccccc agggatgtaa
     2761 ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg
     2821 cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg
     2881 gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg
     2941 gggatacggg gaaaaagctt taggctgaaa gagagattta gaatgacaga atcatagaac
     3001 ggcctgcgta cggatggggc tctagctcta gagcttgata tcgaattcct gcagccccgc
     3061 tagagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg
     3121 gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag
     3181 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg
     3241 aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaagcttt
     3301 aggctgaaag agagatttag aatgacagaa tcatagaacg gcctgggcgc gcctggtggc
     3361 gaacgatcaa cagacgggag aggttacaag tcttcgctga cagcgaccgg actctagggg
     3421 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3481 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3541 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3601 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3661 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3721 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3781 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3841 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt taggctcaat tagtcagcaa
     3901 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     3961 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     4021 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     4081 tcccgggagc ttgtatatcc attttcggag gttgggcttc ggaatcgttt tccgggacgc
     4141 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     4201 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4261 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4321 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     4381 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4441 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4501 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4561 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4621 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4681 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4741 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4801 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4861 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4921 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4981 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5041 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5101 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5161 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5221 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5281 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5341 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5401 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5461 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5521 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5581 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5641 tggccccagt gctgcaatga taccgcgaga tccacgctca ccggctccag atttatcagc
     5701 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5761 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5821 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5881 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5941 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6001 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6061 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6121 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6181 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6241 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6301 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6361 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6421 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6481 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
