LOCUS       sequence-201242-        7870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  human codon optimized TtAgo; pCDNA3 vector backbone, mammalian 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7870)
  AUTHORS   O'Geen H, Ren C, Coggins NB, Bates SL, Segal DJ
  TITLE     Unexpected binding behaviors of bacterial Argonautes in human cells 
            cast doubts on their use as targetable gene regulators.
  JOURNAL   PLoS One. 2018 Mar 27;13(3):e0193818. doi: 
            10.1371/journal.pone.0193818. eCollection 2018.
  PUBMED    29584750
REFERENCE   2  (bases 1 to 7870)
  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: "PLoS One.";
            date: "2018-03-27"; volume: "13(3):e0193818. doi"; pages: " 
            10.1371/journal.pone.0193818. eCollection 2018"
COMMENT     Sequence Label: pCDNA-hTtAgo
FEATURES             Location/Qualifiers
     source          1..7870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        75..454
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        455..658
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     609..629
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     655..679
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             776..796
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             815..880
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3095..3142
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3161..3217
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(3281..3300)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3325..3373
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3575..4003
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3662..3681)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3872..3893
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4012..4032)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4017..4346
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4197..4332
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4259..4278
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4413..5207
                     /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(4467..4486)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5077..5096
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5383..5504
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5420..5439)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5474..5493
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5553..5569)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5553..5569)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5566..5588)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5577..5593
                     /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(5601..5607,5608..5625,5626..5631))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5646..5667
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5784..5801)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5955..6543)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6035..6054)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6714..7505,7506..7574))
                     /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     7337..7356
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7575..7679)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7754..7773)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
       61 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      121 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      181 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      241 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      301 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      361 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      421 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      481 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      541 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      601 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctcg
      661 tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag
      721 acaccgggac cgatccagcc tccggactct agaggatcga acccttgcca ccatgcccaa
      781 gaagaagagg aaggtgggag gctccggagg aagcgactat aaggaccacg acggagacta
      841 caaggatcat gatattgatt acaaagacga tgacgataag ggtggcgggt ccggcggtgg
      901 atccggtacc ggagggagtg gtggaagcgg cggttctggt ggctcaggcg gcagcggccg
      961 gccaatgaat cacctgggaa agaccgaggt gttcctgaac agattcgccc tgcggcccct
     1021 gaaccccgag gaactgaggc cttggagact ggaagtggtg ctggaccctc cacccggcag
     1081 agaagaggtg taccctctgc tggcccaggt ggcaagacgg gctggcggag tgacagtgcg
     1141 gatgggagat ggactggcca gctggtcccc acctgaagtg ctggtgctgg aaggcaccct
     1201 ggccagaatg ggccagacct acgcctaccg gctgtacccc aagggcagaa ggcccctgga
     1261 ccctaaggat cccggcgaga gatctgtgct gagcgccctg gctagacggc tgctgcagga
     1321 aagactgcgg cgcctggaag gcgtgtgggt ggaaggactg gccgtgtaca gaagagagca
     1381 cgccagagga cctggctggc gggtgctggg cggagctgtg ctggatctgt gggtgtcaga
     1441 tagcggcgcc ttcctgctgg aagtggaccc cgcctacaga atcctgtgcg agatgagcct
     1501 ggaagcttgg ctggctcagg gccaccccct gcctaaaaga gtgcggaacg cctacgaccg
     1561 gcggacctgg gaactgctga gactgggcga agaggacccc aaagagctgc ctctgcctgg
     1621 cggactgagc ctgctggatt accacgcctc taagggccgg ctgcagggaa gagaaggcgg
     1681 cagagtggct tgggtggccg atcccaagga ccccagaaag cccatccctc acctgaccgg
     1741 actgctggtg cctgtgctga ccctggaaga tctgcacgag gaagagggca gcctggccct
     1801 gtctctgccc tgggaggaaa gacgcagacg gaccagagag atcgccagct ggatcggcag
     1861 acggctggga ctgggaacac ctgaagctgt gcgggcccag gcctacagac tgagcatccc
     1921 taagctgatg gggcggagag ccgtgtccaa acctgccgat gctctgagag tgggcttcta
     1981 cagagcccag gaaaccgctc tggccctgct gaggctggat ggcgctcagg gatggcccga
     2041 gtttctgaga agggctctgc tgagagcctt cggcgcctct ggcgcttctc tgagactgca
     2101 cacactgcac gcccacccta gtcagggcct ggcctttaga gaggccctgc ggaaggccaa
     2161 agaagagggg gtgcaggctg tgctggtgct gacacctcct atggcctggg aagatcggaa
     2221 ccggctgaaa gccctgctgc tgagggaagg cctgcccagc cagatcctga acgtgcccct
     2281 gcgggaagag gaacggcaca gatgggagaa tgccctgctg ggcctgctgg ccaaagctgg
     2341 actgcaggtg gtggctctgt ctggcgccta tccagctgag ctggccgtgg gctttgatgc
     2401 cggcggaaga gagagcttca gattcggcgg agccgcctgt gcagtgggcg gagatggcgg
     2461 acatctgctg tggacactgc ctgaagccca ggccggcgaa agaatccccc aggaagtcgt
     2521 gtgggatctg ctggaagaga cactgtgggc cttcagacgg aaggccggca gactgcctag
     2581 cagagtgctg ctgctgcgcg acggaagagt gccccaggat gaattcgcac tggccctgga
     2641 agccctggcc cgggaaggca ttgcctacga tctggtgtcc gtgcggaagt ccggcggagg
     2701 cagagtgtat cctgtgcagg gcagactggc tgacggcctg tatgtgcctc tggaagataa
     2761 gacctttctg ctgctgaccg tgcaccggga cttcagaggc acccccagac ctctgaagct
     2821 ggtgcacgaa gccggcgaca caccactgga agctctggca caccagattt tccacctgac
     2881 ccgcctgtac cccgccagcg gctttgcctt tcctagactg cctgcccctc tgcacctggc
     2941 cgaccggctc gtgaaagaag tgggcaggct gggcatccgg cacctgaaag aggtggaccg
     3001 cgagaagctg ttcttcgtgg gaggctctgg cggatccggg ggatctggcg gaagcggagg
     3061 atctgctagc ggtggaggct ccggaggtgg atctaaaagg ccggcggcca cgaaaaaggc
     3121 cggtcaggca aaaaagaaaa agggtggtag tggaagcgga gctactaact tcagcctgct
     3181 gaagcaggct ggagacgtgg aggagaaccc tggacctgcg gccgcatgaa agggttcgat
     3241 ccctaccggt tagtaatgag tttaaacggg ggaggctaac tgaaacacgg aaggagacaa
     3301 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacgggtgtt
     3361 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     3421 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     3481 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagca
     3541 gatctgcgca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     3601 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3661 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3721 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3781 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3841 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3901 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3961 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     4021 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4081 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     4141 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     4201 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4261 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     4321 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     4381 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     4441 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     4501 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     4561 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     4621 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     4681 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     4741 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     4801 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4861 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     4921 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4981 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     5041 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     5101 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     5161 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     5221 gttcgcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     5281 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     5341 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     5401 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5461 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     5521 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5581 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5641 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5701 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5761 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5821 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5881 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5941 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     6001 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     6061 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     6121 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     6181 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6241 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6301 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6361 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6421 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6481 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6541 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6601 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6661 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6721 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6781 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6841 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6901 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6961 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     7021 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     7081 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     7141 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     7201 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7261 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7321 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7381 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7441 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7501 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7561 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7621 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7681 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
     7741 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     7801 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
     7861 gcttgaccga
//
