LOCUS       sequence-174396-        8575 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8575)
  AUTHORS   Arking DE, Krebsova A, Macek M, Macek M, Arking A, Mian IS, Fried L,
            Hamosh A, Dey S, McIntosh I, Dietz HC
  TITLE     Association of human aging with a functional variant of klotho.
  JOURNAL   Proc Natl Acad Sci U S A. 2002 Jan 22. 99(2):856-61.
  PUBMED    11792841
REFERENCE   2  (bases 1 to 8575)
  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. 2002 Jan 22. 99(2):856-61."
COMMENT     Sequence Label: Klotho (membrane)
FEATURES             Location/Qualifiers
     source          1..8575
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        137..516
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        517..720
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     671..691
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     765..784
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        765..783
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3975..4016
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4026..4043
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4066..4083)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4072..4296
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4342..4770
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4429..4448)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4639..4660
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4779..4799)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4784..5113
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4964..5099
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5026..5045
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5180..5974
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5234..5253)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5844..5863
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6150..6271
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6187..6206)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6241..6260
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6320..6336)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6320..6336)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6333..6355)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6344..6360
                     /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)."
     promoter        complement(join(6368..6374,6375..6392,6393..6398))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6413..6434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6551..6568)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6722..7310)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6802..6821)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7481..8272,8273..8341))
                     /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     8104..8123
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8342..8446)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8521..8540)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
       61 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
      121 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
      181 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      241 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      301 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      361 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      421 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      481 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      541 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      601 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      661 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
      721 ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat
      781 agggagaccc aagctggcta gttaagcttg gtaccgagct cggatccact agtccagtgt
      841 ggtggaattg cccttcctgg ctcccgcgta gcatgcccgc cagcgccccg ccgcgccgcc
      901 cgcggccgcc gccgccgtcg ctgtcgctgc tgctggtgct gctgggcctg ggcggccgcc
      961 gcctgcgtgc ggagccgggc gacggcgcgc agacctgggc ccgtttctcg cggcctcctg
     1021 cccccgaggc cgcgggcctc ttccagggca ccttccccga cggcttcctc tgggccgtgg
     1081 gcagcgccgc ctaccagacc gagggcggct ggcagcagca cggcaagggt gcgtccatct
     1141 gggacacgtt cacccaccac cccctggcac cctcgggaga ctcccggaac gccagtctgc
     1201 cgttgggcgc cccgtcgccg ctgcagcccg ccaccgggga cgtagccagc gacagctaca
     1261 acaacgtctt ccgcgacacg gaggcgctgc gcgagctcgg ggtcactcac taccgcttct
     1321 ccatctcgtg ggcgcgagtg ctccccaatg gcagcgcggg cgtccccaac cgcgaggggc
     1381 tgcgctacta ccggcgcctg ctggagcggc tgcgggagct gggcgtgcag cccgtggtca
     1441 ccctgtacca ctgggacctg ccccagcgcc tgcaggacgc ctatggcggc tgggccaacc
     1501 gcgccctggc cgaccacttc agggattacg cggagctctg cttccgccac ttcggcggtc
     1561 aggtcaagta ctggatcacc atcgacaacc cctacgtggt ggcctggcac ggctacgcca
     1621 ccgggcgcct ggcccccggc atccggggca gcccgcggct cgggtacctg gtggcgcaca
     1681 acctcctcct ggctcatgcc aaagtctggc atctctacaa tacttctttc cgtcccactc
     1741 agggaggtca ggtgtccatt gccctaagct ctcactggat caatcctcga agaatgaccg
     1801 accacagcat caaagaatgt caaaaatctc tggactttgt actaggttgg tttgccaaac
     1861 ccgtatttat tgatggtgac tatcccgaga gcatgaagaa taacctttca tctattctgc
     1921 ctgattttac tgaatctgag aaaaagttca tcaaaggaac tgctgacttt tttgctcttt
     1981 gctttggacc caccttgagt tttcaacttt tggaccctca catgaagttc cgccaattgg
     2041 aatctcccaa cctgaggcaa ctgctttcct ggattgacct tgaatttaac catcctcaaa
     2101 tatttattgt ggaaaatggc tggtttgtct cagggaccac caagagagat gatgccaaat
     2161 atatgtatta cctcaaaaag ttcatcatgg aaaccttaaa agccatcaag ctggatgggg
     2221 tggatgtcat cgggtatacc gcatggtccc tcatggatgg tttcgagtgg cacagaggtt
     2281 acagcatcag gcgtggactc ttctatgttg actttctaag ccaggacaag atgttgttgc
     2341 caaagtcttc agccttgttc taccaaaagc tgatagagaa aaatggcttc cctcctttac
     2401 ctgaaaatca gcccctagaa gggacatttc cctgtgactt tgcttgggga gttgttgaca
     2461 actacattca agtagatacc actctgtctc agtttaccga cctgaatgtt tacctgtggg
     2521 atgtccacca cagtaaaagg cttattaaag tggatggggt tgtgaccaag aagaggaaat
     2581 cctactgtgt tgactttgct gccatccagc cccagatcgc tttactccag gaaatgcacg
     2641 ttacacattt tcgcttctcc ctggactggg ccctgattct ccctctgggt aaccagtccc
     2701 aggtgaacca caccatcctg cagtactatc gctgcatggc cagcgagctt gtccgtgtca
     2761 acatcacccc agtggtggcc ctgtggcagc ctatggcccc gaaccaagga ctgccgcgcc
     2821 tcctggccag gcagggcgcc tgggagaacc cctacactgc cctggccttt gcagagtatg
     2881 cccgactgtg ctttcaagag ctcggccatc acgtcaagct ttggataacg atgaatgagc
     2941 cgtatacaag gaatatgaca tacagtgctg gccacaacct tctgaaggcc catgccctgg
     3001 cttggcatgt gtacaatgaa aagtttaggc atgctcagaa tgggaaaata tccatagcct
     3061 tgcaggctga ttggatagaa cctgcctgcc ctttctccca aaaggacaaa gaggtggctg
     3121 agagagtttt ggaatttgac attggctggc tggctgagcc cattttcggc tctggagatt
     3181 atccatgggt gatgagggac tggctgaacc aaagaaacaa ttttcttctt ccttatttca
     3241 ctgaagatga aaaaaagcta atccagggta cctttgactt tttggcttta agccattata
     3301 ccaccatcct tgtagactca gaaaaagaag atccaataaa atacaatgat tacctagaag
     3361 tgcaagaaat gaccgacatc acgtggctca actcccccag tcaggtggcg gtagtgccct
     3421 gggggttgcg caaagtgctg aactggctga agttcaagta cggagacctc cccatgtaca
     3481 taatatccaa cggaatcgat gacgggctgc atgctgagga cgaccagctg agggtgtatt
     3541 atatgcagaa ttacataaac gaagctctca aagcccacat actggatggt atcaatcttt
     3601 gcggatactt tgcttattcg tttaacgacc gcacagctcc gaggtttggc ctctatcgtt
     3661 atgctgcaga tcagtttgag cccaaggcat ccatgaaaca ttacaggaaa attattgaca
     3721 gcaatggttt cccgggccca gaaactctgg aaagattttg tccagaagaa ttcaccgtgt
     3781 gtactgagtg cagttttttt cacacccgaa agtctttact ggctttcata gcttttctat
     3841 tttttgcttc tattatttct ctctccctta tattttacta ctcgaagaaa ggcagaagaa
     3901 gttacaaaaa gggcaattct gcagatatcc agcacagtgg cggccgctcg agtctagagg
     3961 gcccgcggtt cgaaggtaag cctatcccta accctctcct cggtctcgat tctacgcgta
     4021 ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt
     4081 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     4141 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     4201 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     4261 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     4321 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4381 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4441 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4501 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     4561 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4621 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     4681 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4741 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     4801 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4861 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4921 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     4981 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     5041 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     5101 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     5161 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     5221 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     5281 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     5341 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     5401 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     5461 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     5521 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     5581 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     5641 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     5701 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     5761 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     5821 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     5881 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     5941 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgcgaaatga
     6001 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     6061 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     6121 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     6181 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     6241 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     6301 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     6361 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     6421 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     6481 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     6541 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     6601 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     6661 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     6721 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6781 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6841 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6901 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6961 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     7021 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     7081 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     7141 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     7201 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     7261 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     7321 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     7381 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     7441 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     7501 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     7561 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     7621 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     7681 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     7741 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     7801 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7861 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7921 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7981 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     8041 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     8101 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     8161 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     8221 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     8281 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     8341 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     8401 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     8461 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     8521 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtg
//
