LOCUS       sequence-392290-        7372 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable and dox. inducible expression of Trim69 isoform E (codon 
            optimized) after retroviral-mediated gene transfer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7372)
  AUTHORS   Song Y, Nguyen XN, Kumar A, da Silva C, Picard L, Etienne L, 
            Cimarelli A
  TITLE     Trim69 is a microtubule regulator that acts as a pantropic viral 
            inhibitor.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Oct 25;119(43):e2211467119. doi: 
            10.1073/pnas.2211467119. Epub 2022 Oct 17.
  PUBMED    36251989
REFERENCE   2  (bases 1 to 7372)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-10-25"; volume: "119(43):e2211467119. 
            doi"; pages: " 10.1073/pnas.2211467119. Epub 2022 Oct 17"
COMMENT     Sequence Label: pRetroX-Tight-Pur-Trim69 isoform E (codon opti...
FEATURES             Location/Qualifiers
     source          1..7372
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        73..452
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        453..656
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             657..832
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    895..1094
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1295..1711
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1641..1663
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1679..1695
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1747..2061
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1755..1773
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1791..1809
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1826..1844
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1862..1880
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1898..1916
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1933..1951
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1969..1987
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2034..2058
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2081..2104
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        2922..3421
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2957..2980)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3354..3373
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3442..4041
                     /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(3442..3461)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3938..3958
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4222..4647
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(4750..4772)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4908..4928)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4913..5242
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5093..5228
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5264..5283
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5364..5381)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5535..6123)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5615..5634)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6294..7085,7086..7154))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6917..6936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7155..7259)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7327..7345
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cattgcatac gttgtatcca tatcataata tgtacattta tattggctca tgtccaacat
       61 taccgccatg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      121 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      181 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      241 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      301 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      361 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      421 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      481 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      541 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      601 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcaat
      661 aaaagagccc acaacccctc actcggcgcg ccagtcttcc gatagactgc gtcgcccggg
      721 tacccgtatt cccaataaag cctcttgctg tttgcatccg aatcgtggtc tcgctgttcc
      781 ttgggagggt ctcctctgag tgattgacta cccacgacgg gggtctttca tttgggggct
      841 cgtccgggat ttggagaccc ctgcccaggg accaccgacc caccaccggg aggtaagctg
      901 gccagcaact tatctgtgtc tgtccgattg tctagtgtct atgtttgatg ttatgcgcct
      961 gcgtctgtac tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga
     1021 gttctgaaca cccggccgca accctgggag acgtcccagg gactttgggg gccgtttttg
     1081 tggcccgacc tgaggaaggg agtcgatgtg gaatccgacc ccgtcaggat atgtggttct
     1141 ggtaggagac gagaacctaa aacagttccc gcctccgtct gaatttttgc tttcggtttg
     1201 gaaccgaagc cgcgcgtctt gtctgctgca gcgctgcagc atcgttctgt gttgtctctg
     1261 tctgactgtg tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag
     1321 tttgacctta ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt
     1381 caagaagaga cgttgggtta ccttctgctc tgcagaatgg ccaaccttta acgtcggatg
     1441 gccgcgagac ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc
     1501 acctggcccg catggacacc cagaccaggt cccctacatc gtgacctggg aagccttggc
     1561 ttttgacccc cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc
     1621 tccatccgcc ccgtctctcc cccttgaacc tcctcgttcg accccgcctc gatcctccct
     1681 ttatccagcc ctcactcctt ctctaggcgc cggaattgaa gatctgaggc cctttcgtct
     1741 tcactcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag
     1801 tgatagagaa cgatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta
     1861 ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt
     1921 atgtcgagtt tatccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg
     1981 atagagaacg tatgtcgagg taggcgtgta cggtgggagg cctatataag cagagctcgt
     2041 ttagtgaacc gtcagatcgc ctggagaagg atccaccatg gactacaaag acgatgacga
     2101 taagggtggc ggagcggccg ccgaggagga gctggccatt cagcagggcc agctggagac
     2161 caccctgaag gagctgcaaa ccctcaggaa catgcagaag gaggccatcg ccgctcacaa
     2221 ggatattacc accctgctgc actccctgga gcagggcatg aaggtcctgg ccaccagaga
     2281 gctcatttcc aggaagctga acctgggcca gtataagggc cccattcagt acatggtgtg
     2341 gagggagatg caggacaccc tgtgtcccgg cctgagccct ctgacactgg accccaaaac
     2401 agcccacccc aatctggtgc tgtccaagag ccagacctcc gtgtggcatg gcgacatcaa
     2461 gaagatcatg cccgatgacc ccgagaggtt tgacagcagc gtcgctgtgc tgggctccag
     2521 aggctttacc agcggcaaat ggtactggga agtggaggtg gccaaaaaga ccaaatggac
     2581 agtgggagtg gtgagggaaa gcattatcag gaagggcagc tgtcctctga cacctgagca
     2641 gggcttttgg ctgctgaggc tgagaaacca gacagacctg aaggccctgg acctccccag
     2701 cttcagcctg accctgacaa acaacctgga caaggtcggc atctacctgg actacgaggg
     2761 aggccagctg agcttctata atgccaaaac catgacacac atctatacct tttccaacac
     2821 atttatggag aagctgtacc cttatttctg tccctgcctg aacgacggcg gcgaaaataa
     2881 ggagcccctg cacattctgc acccccagtg agaattctac cgggtagggg aggcgctttt
     2941 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag
     3001 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     3061 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc
     3121 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     3181 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     3241 gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct
     3301 caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg
     3361 cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc
     3421 gacctgcagc ccaagcttac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     3481 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     3541 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     3601 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     3661 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     3721 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     3781 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     3841 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     3901 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     3961 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     4021 acccgcaagc ccggtgcctg acgcccgccc cacgacccgc agcgcccgac cgaaaggagc
     4081 gcacgacccc atgcatcggc gtctcgagat atcagtggtc caggctctag ttttgactca
     4141 acaatatcac cagctgaagc ctatagagta cgagccatag ataaaataaa agattttatt
     4201 tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca agctagctta
     4261 agtaacgcca ttttgcaagg catggaaaaa tacataactg agaatagaga agttcagatc
     4321 aaggtcagga acagatggaa cagggtcgac cctagagaac catcagatgt ttccagggtg
     4381 ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg
     4441 cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg
     4501 gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct
     4561 tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg
     4621 actacccgtc agcgggggtc tttcatttgg gggctcgtcc gggatcggga gacccctgcc
     4681 cagggaccac cgacccacca ccgggaggta agctggctgc ctcgcgcgtt tcggtgatga
     4741 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     4801 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     4861 agccatgacc cagtcacgta gcgatagcgg agtgtagatc cggctgtgga atgtgtgtca
     4921 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4981 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     5041 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     5101 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     5161 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     5221 tggaggccta ggcttttgca aaaagcttac tggcttaact atgcggcatc agagcagatt
     5281 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     5341 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5401 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5461 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5521 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5581 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5641 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5701 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5761 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5821 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5881 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5941 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     6001 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6061 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6121 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6181 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6241 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6301 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6361 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6421 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6481 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6541 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6601 gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6661 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6721 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6781 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6841 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6901 ccggcgtcaa cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6961 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7021 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7081 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7141 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7201 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7261 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     7321 tataaaaata ggcgtatcac gaggcccttt cgtcttcaag aattagcttg gc
//
