LOCUS       sequence-318335-        8223 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human codon optimized HSD17B10. Expression is controlled by 
            doxycycline..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8223)
  AUTHORS   Condon KJ, Orozco JM, Adelmann CH, Spinelli JB, van der Helm PW, 
            Roberts JM, Kunchok T, Sabatini DM
  TITLE     Genome-wide CRISPR screens reveal multitiered mechanisms through 
            which mTORC1 senses mitochondrial dysfunction.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Jan 26;118(4). pii: 2022120118. doi: 
            10.1073/pnas.2022120118.
  PUBMED    33483422
REFERENCE   2  (bases 1 to 8223)
  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: "2021-01-26"; volume: "118(4). pii: 
            2022120118. doi"; pages: " 10.1073/pnas.2022120118"
COMMENT     Sequence Label: pCW57.kc1-HSD17B10
FEATURES             Location/Qualifiers
     source          1..8223
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      40..175
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        175..246
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(247..315,316..1107)
                     /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(465..484)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1278..1866
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1767..1786
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2020..2037
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2154..2175
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2190..2195,2196..2213,2214..2220)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2228..2244
                     /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)."
     primer_bind     2233..2255
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2252..2268
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2252..2268
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2287..2307
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2289..2307
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2335..2561
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2562..2742
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2789..2914
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3407..3640
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3825..3869
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    4082..4199
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    4263..4281
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4303..4321
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4368..4392
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5329..5394
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        5401..5911
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     5830..5848
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             5921..6313
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MVPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             6323..6376
                     /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             join(6377..6994,6995..7000,7001..7036,7037..7039,7040..7075,
                     7076..7078,7079..7114,7115..7123)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=tTA-Advanced
                     /note="In the Tet-Off(R) system, tTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(7163..7183)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7581..7592)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7797..8030
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8102..8223
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8139..8158)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8193..8212
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc
       61 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
      121 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctttca
      181 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
      241 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
      301 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
      361 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
      421 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
      481 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
      541 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
      601 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
      661 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
      721 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
      781 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
      841 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
      901 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
      961 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     1021 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     1081 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     1141 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     1201 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     1261 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1321 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1381 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     1441 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1501 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     1561 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1621 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     1681 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     1741 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     1801 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     1861 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     1921 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     1981 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2041 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     2101 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     2161 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     2221 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     2281 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
     2341 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
     2401 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
     2461 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
     2521 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
     2581 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     2641 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     2701 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     2761 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     2821 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     2881 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     2941 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     3001 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     3061 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     3121 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     3181 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     3241 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     3301 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     3361 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     3421 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     3481 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     3541 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     3601 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     3661 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     3721 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     3781 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     3841 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     3901 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     3961 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     4021 acctcccaac cccgagggga caattctcga cctcgagaca aatggcagta ttcatccaca
     4081 attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     4141 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc
     4201 gggtttatta cagggacagc agagatccac tttggccgcg aatcgacgta tctacagttt
     4261 actccctatc agtgatagag attacgtata tccagtttac tttccctatc agtgatagag
     4321 attacgtata agctttaggc gtgtacggtg ggcgcctata aaagcagagc tcgtttagtg
     4381 aaccgtcaga tcgcctggag caattccaca acacttttgt cttataccaa ctttccgtac
     4441 cacttcctac cctcgtaaag tcagttggcc aggcctagtt ggccaggcct agtgccgcca
     4501 ccgtcgacga tggcggctgc ctgtaggagc gtgaagggcc tcgtagctgt tattaccggg
     4561 ggcgcctctg gtctcggtct cgctacggca gagcgacttg taggacaggg ggcatctgcc
     4621 gtattgctcg accttcctaa ttcaggtggt gaggcccaag cgaaaaaact gggtaataac
     4681 tgcgtattcg caccggctga cgtgacctct gaaaaagatg tgcaaacagc actggcgctg
     4741 gctaaaggaa aattcggccg agtggacgtt gctgtaaact gcgccggcat tgcggtcgca
     4801 agcaagactt acaacctcaa gaaaggccag acccacacgt tggaagactt ccagagggta
     4861 ctcgacgtaa acctgatggg tacattcaat gtaatacgcc tcgtagccgg cgaaatgggt
     4921 caaaacgagc cggatcaggg tggacaacgc ggggtcatta ttaacacggc ttccgtcgcg
     4981 gcatttgaag gacaggtggg acaagcagca tactctgctt ccaagggagg catcgttggt
     5041 atgacattgc ctatcgcacg ggacctggcc ccgataggga taagggtaat gacgattgca
     5101 ccgggccttt tcggaacccc gctgctcact agtctcccag agaaagtgtg caacttcctt
     5161 gcaagccagg ttccattccc gtcacgactc ggagaccctg ccgagtatgc acaccttgta
     5221 caagctataa ttgagaatcc tttcctgaac ggggaagtta ttcgacttga tggagcaata
     5281 agaatgcagc cgtaaggatc cacgtcgggt ggtggtggtg ggaatctcga ctacaaagac
     5341 catgacggtg attataaaga tcatgacatc gattacaagg atgacgatga caaggattga
     5401 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc
     5461 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc
     5521 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc
     5581 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac
     5641 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc
     5701 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag
     5761 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct
     5821 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct
     5881 cgttgaccga atcaccgacc tctctcccca gcaattcacc atggttcctt tgtctcaaga
     5941 agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga
     6001 ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt
     6061 atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc
     6121 ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag
     6181 cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt
     6241 gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta
     6301 tgtgtgggag ggccccggtg ccgaaggtag aggttctctc ctcacttgtg gtgatgttga
     6361 agaaaaccct ggtccaatgt ctagactgga caagagcaaa gtcataaact ctgctctgga
     6421 attactcaat gaagtcggta tcgaaggcct gacgacaagg aaactcgctc aaaagctggg
     6481 agttgagcag cctaccctgt actggcacgt gaagaacaag cgggccctgc tcgatgccct
     6541 ggcaatcgag atgctggaca ggcatcatac ccacttctgc cccctggaag gcgagtcatg
     6601 gcaagacttt ctgcggaaca acgccaagtc attccgctgt gctctcctct cacatcgcga
     6661 cggggctaaa gtgcatctcg gcacccgccc aacagagaaa cagtacgaaa ccctggaaaa
     6721 tcagctcgcg ttcctgtgtc agcaaggctt ctccctggag aacgcactgt acgctctgtc
     6781 cgccgtgggc cactttacac tgggctgcgt attggaggat caggagcatc aagtagcaaa
     6841 agaggaaaga gagacaccta ccaccgattc tatgccccca cttctgagac aagcaattga
     6901 gctgttcgac catcagggag ccgaacctgc cttccttttc ggcctggaac taatcatatg
     6961 tggcctggag aaacagctaa agtgcgaaag cggcgggccg gccgacgccc ttgacgattt
     7021 tgacttagac atgctcccag ccgatgccct tgacgacttt gaccttgata tgctgcctgc
     7081 tgacgctctt gacgattttg accttgacat gctccccggg taactaagta aggatcgatc
     7141 caagatatcg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     7201 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     7261 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     7321 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     7381 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     7441 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     7501 ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     7561 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     7621 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     7681 tttgggccgc ctccccgcct gtttcgcctc ggcgtccgga ctagaggtac ctttaagacc
     7741 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     7801 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg
     7861 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     7921 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     7981 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc
     8041 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     8101 gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     8161 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     8221 tta
//
