LOCUS       sequence-174787-        6050 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of C12orf66.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6050)
  AUTHORS   Wolfson RL, Chantranupong L, Wyant GA, Gu X, Orozco JM, Shen K, 
            Condon KJ, Petri S, Kedir J, Scaria SM, Abu-Remaileh M, Frankel WN, 
            Sabatini DM
  TITLE     KICSTOR recruits GATOR1 to the lysosome and is necessary for 
            nutrients to regulate mTORC1.
  JOURNAL   Nature. 2017 Feb 15. doi: 10.1038/nature21423.
  PUBMED    28199306
REFERENCE   2  (bases 1 to 6050)
  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: "Nature. 
            2017 Feb 15. doi: 10.1038/nature21423."
COMMENT     Sequence Label: pRK5-FLAG-C12orf66
FEATURES             Location/Qualifiers
     source          1..6050
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             924..947
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2337..2346
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2350..2484
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2387..2406)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2441..2460
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2553..2910
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2553..2744
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2576..2596)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2761..2896
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2930..2947)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2930..2946)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2939..2961)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3159..3614
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3246..3265)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3456..3477
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3631..3650)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3750..3772
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3810..3828)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3896..4000
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4001..4069,4070..4861)
                     /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(4219..4238)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5032..5620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5521..5540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5774..5791
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5908..5929
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5944..5949,5950..5967,5968..5974)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5982..5998
                     /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     5987..6009
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6006..6022
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6006..6022
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgattgaatt caccatggaa atggactaca aggatgacga tgacaagggt ggtggtggtg
      961 gttcgacgat gggggagtct atcccgctgg ccgccccggt cccggtggaa caggcggtgc
     1021 tggagacgtt cttctctcac ctgggtatct tctcttacga caaggctaag gacaatgtgg
     1081 agaaggaacg agaggccaac aagagcgcgg ggggcagctg gctgtcgctg ctggcggcct
     1141 tggcgcacct ggccgcggcc gagaaggtct atcacagcct cacctacctg gggcagaaac
     1201 tagggggcca gtctttcttc agcaggaagg attccatccg caccatctat acttcattgc
     1261 ataatgagct gaagaaggtg gtgactggcc gtggtgccct gggtgggact gctcctcacg
     1321 tggaagaact cctttcccac ctgtcagagc agctctgctt ctttgttcag gctcggatgg
     1381 agatagcaga cttctatgag aagatgtaca ccctcagcac acagaagttc atcaatgctg
     1441 aagagctcgt tggccttttg gatgccatca tgaaaaaata cagctccaga tttcaccacc
     1501 caatcctcag tcctttggaa agcagtttcc agctggaagt tgacgtcctg tgtcatctcc
     1561 tgaaagccca ggcccaggtc tcagagtgga agttcctccc atctctggtt aatttacaca
     1621 gtgcgcacac caaactgcag acgtggggcc agatctttga gaaacagcgg gagaccaaga
     1681 aacatctgtt tggagggcag tctcagaagg ccgtgcagcc tccacacctt ttcctttggc
     1741 tgatgaaact caaaaacatg cttcttgcca agtttagctt ttactttcat gaggctctga
     1801 gccgacaaac gactgcttca gaaatgaaaa cgttgacagc aaaagctaac ccagactttt
     1861 ttggaaagat ttccagcttc atcagaaaat atgatgctgc caatgtgtcc ttaatttttg
     1921 acaacagagg ttctgagagt tttcagggtc atggttatca ccacccccat tcctacagag
     1981 aggcccccaa gggtgtggac cagtatccag ctgtagtgtc tctgcccagc gacaggccag
     2041 tcatgcactg gcccaatgtc atcatgatca tgacggaccg cacatctgat ctgaacagct
     2101 tggagaaagt ggtccacttc tatgatgaca aagttcagag tacctacttc ctaacccgcc
     2161 cggaacctca ctttaccatt gtcatcattt ttgagtcaaa gaaatctgag agagactccc
     2221 actttatttc cttcctcaat gaggtctcac ttgcccttaa gaaccccaaa gtgtttgcaa
     2281 gtctgaaacc tggatccaaa ggttaggcgg ccaagtaagt aaggatcccc agcttggccg
     2341 ccatggccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     2401 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     2461 aatgtatctt atcatgtctg gatcgggaat taattcggcg cagcaccatg gcctgaaata
     2521 acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat
     2581 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2641 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2701 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     2761 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     2821 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     2881 ggcttttttg gaggcctagg cttttgcaaa aagctgttaa cagcttggca ctggccgtcg
     2941 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     3001 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     3061 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     3121 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     3181 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3241 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3301 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3361 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3421 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3481 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc
     3541 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     3601 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3661 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3721 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3781 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     3841 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     3901 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3961 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4021 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4081 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4141 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4201 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     4261 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     4321 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     4381 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     4441 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     4501 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     4561 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     4621 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4681 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4741 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4801 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4861 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4921 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4981 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5041 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5101 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5161 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5221 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5281 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5341 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5401 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5461 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5521 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5581 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5641 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5701 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5761 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     5821 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     5881 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     5941 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     6001 tcacacagga aacagctatg acatgattac gaattaattc gagctcgccc
//
