LOCUS       sequence-328546-        5649 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the Tet3G transactivator and puromycin resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5649)
  AUTHORS   Michalec-Wawiorka B, Czapinski J, Filipek K, Rulak P, Czerwonka A, 
            Tchorzewski M, Rivero-Muller A
  TITLE     An Improved Vector System for Homogeneous and Stable Gene 
            Regulation.
  JOURNAL   Int J Mol Sci. 2021 May 14;22(10). pii: ijms22105206. doi: 
            10.3390/ijms22105206.
  PUBMED    34069024
REFERENCE   2  (bases 1 to 5649)
  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: "Int J Mol 
            Sci."; date: "2021-05-14"; volume: "22(10). pii: ijms22105206. doi";
            pages: " 10.3390/ijms22105206"
COMMENT     Sequence Label: pCAG-TET3G-T2A-Puro
FEATURES             Location/Qualifiers
     source          1..5649
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1250..1993
                     /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"
     CDS             2009..2062
                     /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             2063..2662
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2063..2082)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2559..2579
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2690..2707)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2696..2903
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2912..3052
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2912..3041
                     /label=AAV2 ITR
     rep_origin      3127..3582
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3214..3233)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3424..3445
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3599..3618)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3718..3740
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3778..3796)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3864..3968
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3969..4037,4038..4829)
                     /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(4187..4206)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5000..5588
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5489..5508
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccgggg gggggggggg ggggsggggg gsggggsggg gsgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
     1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tgtctagact
     1261 ggacaagagc aaagtcataa actctgctct ggaattactc aatggagtcg gtatcgaagg
     1321 cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc tgtactggca
     1381 cgtgaagaac aagcgggccc tgctcgatgc cctgccaatc gagatgctgg acaggcatca
     1441 tacccactcc tgccccctgg aaggcgagtc atggcaagac tttctgcgga acaacgccaa
     1501 gtcataccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc tcggcacccg
     1561 cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt gtcagcaagg
     1621 cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta cactgggctg
     1681 cgtattggag gaacaggagc atcaagtagc aaaagaggaa agagagacac ctaccaccga
     1741 ttctatgccc ccacttctga aacaagcaat tgagctgttc gaccggcagg gagccgaacc
     1801 tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc taaagtgcga
     1861 aagcggcggg ccgaccgacg cccttgacga ttttgactta gacatgctcc cagccgatgc
     1921 ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt ttgaccttga
     1981 catgctcccc ggggaattcg gcagtggaga gggcagagga agtctgctaa catgcggtga
     2041 cgtcgaggag aatcctggcc caatgaccga gtacaagccc acggtgcgcc tcgccacccg
     2101 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     2161 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     2221 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     2281 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     2341 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     2401 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga
     2461 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     2521 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     2581 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     2641 gacccgcaag cccggtgcct gagaattcta actagagctc gctgatcagc ctcgactgtg
     2701 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2761 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2821 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2881 gagaatagca ggcatgctgg ggagcggccg caggaacccc tagtgatgga gttggccact
     2941 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     3001 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     3061 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     3121 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3181 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     3241 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3301 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     3361 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3421 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     3481 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     3541 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     3601 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     3661 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3721 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     3781 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     3841 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3901 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3961 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4021 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     4081 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     4141 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     4201 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     4261 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     4321 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     4381 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4441 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4501 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4561 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4621 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4681 gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4741 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4801 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4861 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4921 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4981 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5041 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5101 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     5161 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5221 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5281 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5341 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5401 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5461 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5521 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5581 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5641 ctcacatgt
//
