LOCUS       sequence-174789-        6100 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 6100)
  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 6100)
  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-HA-C12orf66
FEATURES             Location/Qualifiers
     source          1..6100
                     /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"
     regulatory      950..959
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             962..988
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2387..2396
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2400..2534
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2437..2456)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2491..2510
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2603..2960
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2603..2794
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2626..2646)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2811..2946
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2980..2997)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2980..2996)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2989..3011)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3209..3664
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3296..3315)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3506..3527
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3681..3700)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3800..3822
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3860..3878)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3946..4050
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4051..4119,4120..4911)
                     /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(4269..4288)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5082..5670
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5571..5590
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5824..5841
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5958..5979
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5994..5999,6000..6017,6018..6024)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6032..6048
                     /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     6037..6059
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6056..6072
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6056..6072
                     /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 cccgaaccga cagtcggtct cttcaccaag gccattcgcg ccaccatggg
      961 ctacccctat gatgtgcctg actacgcaga tctcaatggt ggtggtggtg ggtcgacgat
     1021 gggggagtct atcccgctgg ccgccccggt cccggtggaa caggcggtgc tggagacgtt
     1081 cttctctcac ctgggtatct tctcttacga caaggctaag gacaatgtgg agaaggaacg
     1141 agaggccaac aagagcgcgg ggggcagctg gctgtcgctg ctggcggcct tggcgcacct
     1201 ggccgcggcc gagaaggtct atcacagcct cacctacctg gggcagaaac tagggggcca
     1261 gtctttcttc agcaggaagg attccatccg caccatctat acttcattgc ataatgagct
     1321 gaagaaggtg gtgactggcc gtggtgccct gggtgggact gctcctcacg tggaagaact
     1381 cctttcccac ctgtcagagc agctctgctt ctttgttcag gctcggatgg agatagcaga
     1441 cttctatgag aagatgtaca ccctcagcac acagaagttc atcaatgctg aagagctcgt
     1501 tggccttttg gatgccatca tgaaaaaata cagctccaga tttcaccacc caatcctcag
     1561 tcctttggaa agcagtttcc agctggaagt tgacgtcctg tgtcatctcc tgaaagccca
     1621 ggcccaggtc tcagagtgga agttcctccc atctctggtt aatttacaca gtgcgcacac
     1681 caaactgcag acgtggggcc agatctttga gaaacagcgg gagaccaaga aacatctgtt
     1741 tggagggcag tctcagaagg ccgtgcagcc tccacacctt ttcctttggc tgatgaaact
     1801 caaaaacatg cttcttgcca agtttagctt ttactttcat gaggctctga gccgacaaac
     1861 gactgcttca gaaatgaaaa cgttgacagc aaaagctaac ccagactttt ttggaaagat
     1921 ttccagcttc atcagaaaat atgatgctgc caatgtgtcc ttaatttttg acaacagagg
     1981 ttctgagagt tttcagggtc atggttatca ccacccccat tcctacagag aggcccccaa
     2041 gggtgtggac cagtatccag ctgtagtgtc tctgcccagc gacaggccag tcatgcactg
     2101 gcccaatgtc atcatgatca tgacggaccg cacatctgat ctgaacagct tggagaaagt
     2161 ggtccacttc tatgatgaca aagttcagag tacctacttc ctaacccgcc cggaacctca
     2221 ctttaccatt gtcatcattt ttgagtcaaa gaaatctgag agagactccc actttatttc
     2281 cttcctcaat gaggtctcac ttgcccttaa gaaccccaaa gtgtttgcaa gtctgaaacc
     2341 tggatccaaa ggttaggcgg ccaagtaagt aaggatcccc agcttggccg ccatggccca
     2401 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2461 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2521 atcatgtctg gatcgggaat taattcggcg cagcaccatg gcctgaaata acctctgaaa
     2581 gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     2641 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2701 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2761 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     2821 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     2881 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     2941 gaggcctagg cttttgcaaa aagctgttaa cagcttggca ctggccgtcg ttttacaacg
     3001 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     3061 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     3121 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3181 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     3241 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3301 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3361 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3421 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3481 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3541 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     3601 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     3661 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     3721 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3781 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3841 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     3901 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3961 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4021 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4081 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4141 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4201 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4261 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4321 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4381 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4441 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4501 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4561 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4621 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4681 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4741 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4801 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4861 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4921 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4981 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5041 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5101 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5161 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5221 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5281 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5341 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5401 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5461 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5521 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5581 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5641 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5701 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5761 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     5821 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     5881 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     5941 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     6001 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     6061 aacagctatg acatgattac gaattaattc gagctcgccc
//
