LOCUS       sequence-194114-        7128 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CLIP-tagged Neurokinin-1 Receptor in Mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7128)
  AUTHORS   Ana Egana
  TITLE     Egana Lab NEB plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7128)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCLIPf-NK1R Control Plasmid
FEATURES             Location/Qualifiers
     source          1..7128
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(91..110)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          93..322
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     CDS             complement(413..442)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             complement(473..496)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1682..2227)
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylcytosine 
                     derivatives"
                     /label=CLIP-tag(TM)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLIQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISESHLAALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLA
                     VKEWLLAHEGHRLGKPGLG"
     primer_bind     complement(2344..2363)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2345..2363)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2408..2611)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2437..2457)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2612..2991
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3163..3182
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3257..3361
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3362..3430,3431..4222)
                     /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(3580..3599)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4393..4981
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4882..4901
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5135..5152
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5269..5290
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5305..5310,5311..5328,5329..5335)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5343..5359
                     /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     5348..5370
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5367..5383
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5367..5383
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5485..5606
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5550..5569
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5736..6539)
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5847..5866)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6457..6476
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     misc_feature    join(6562..6570,6571..6573,6574..7128,1..20)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(6735..6754)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     6964..6981
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
ORIGIN
        1 ggggggggga gggagagggg ggggggggag aggggcggaa ttggccgccc tagatgcatg
       61 ctcgacctgc agttggacct gggagtggac acctgtggag agaaaggcaa agtggatgtc
      121 attgtcactc aagtgtatgg ccagatctca agcctgccac acctcaagct tgacaacaaa
      181 aagattgtct tttctgacca gatggacgcg gccaccctca aaggcatcac cgcgggccag
      241 gtgaatatca aatcctcctc gtttttggaa actgacaatc ttagcgcaga agtcatgccc
      301 gcttttgaga gggagtactc accccaacag ctggccctcg cagacagcga attaattcca
      361 gcacactgga tcagttatct atgcggccgc ctcactcgag tcaggatcct taatggtgat
      421 ggtgatgatg atgatgatga tggctgctgc cgctgccgcg cggcacaccg gtcttgtcat
      481 cgtcgtcctt gtagtcggag agcacattgg aggagaagct gaagctctct gtcatggtct
      541 tggagtcact tcgtgaagag cagttggagg tcaggtccag ggacgagggt gtggccttgg
      601 ggccgtcctc tggctcctcc tcgtgggccc ccaccactgt ggagatggtg gtctccaggc
      661 ggctgacttt gtacacactg ccctgggtct ggagataccg ggtggatttc atttccagcc
      721 cctcatagtc gccggcgctg atgaaggggc agcaccggaa ggcatgcttg aagcccagac
      781 ggaacctgtc attgaggcag cagtagatga tggggttgta catggtggag ctcatggcca
      841 gccacatgat ggccaggtag acctgctgga taaacttctt caggtagaga tctgggttgg
      901 tgtagggcag gaggaagaag atgtggaagg gcagcctgca gatggcgaag gtgcacacca
      961 cgacaatcat cattttgacc accttgcgct tggcagagac ttgctcgtgg tagcggtcag
     1021 aggagtcccc ggggatctca ctggcccata gtgtgattcc cactacggtg tatgcatagc
     1081 caatcaccag cagggggagg aagtagatca gcacagtcac acagatgtgg tacactttct
     1141 cataaatctt gttcggatgc tctggccatt cgatcatgca cacgactctg ctgggcatgg
     1201 tctctgtggt tgagtagtag ccctggggga aggccagcag gagagccagg acccagatga
     1261 cacagatgac cactttggtg gctgtggctg acagccgggg ctggagggga tgtatgatgg
     1321 ccatgtacct atcaaaggcc acagccgtca tggagtagat actggcgaag acagcggcga
     1381 tgggaaagaa gttgtggaac ttgcagtaga acaggccgta gtaccattcg ttgtggacag
     1441 cataggtgaa gttcaccact gtattgaatg cagccatgga ggcctccgcg aaggccaggt
     1501 tcaccagaaa atagttcgtc actgtcctca ttcttttgtg ggctaagatg atccacatca
     1561 ctaccacgtt gcccaccaca gaggtcacca caatgaccgt gtaggcagct gcccaaagga
     1621 caatttgcca ggctggttgc acgaactgat tgggttccga ggtgttagtg gaacctgcag
     1681 gacccagccc aggcttgccc agtctgtggc cctcgtgggc cagcagccac tctttcacgg
     1741 cgagcccgcc caggtagggc cccacgtcgc tgtcgccctg caccacccgg tggcagggga
     1801 tcagaatggg cacgggattt ccgtccaggg cggtgttcac ggcggcggtg gcggcgggat
     1861 tgcccaccag ggcggccagg tggctctcgc tgatgacctc tccgaacttc accactttca
     1921 gcagtttcca cagcacctgg cgggtaaagc tctcctgctg gaacactggg tggtgcaggg
     1981 ctggcacagg gaactcctcg atggcctcag gctggtgaaa gtaggcgttg agccaggcgg
     2041 tggcctggat cagtggctct ggtccgccca gcacggcggc tggggcaggc acttccacgg
     2101 cgtcggcggc agatgttcct ttgcccagga agatgatacg gtgcaggccc tgttcgcacc
     2161 cagacagttc cagcttgccc agagggctat ccagggtggt gcgcttcatt tcgcagtctt
     2221 tgtccatgct agcgctgcct tctcccggcc ctgtcagcat ggaaaggaac aaggccaaca
     2281 gcacctgcgg gatgcagagc cgcatggtga tatcgatccg agctcggtac caagcttggg
     2341 tctccctata gtgagtcgta ttaatttcga taagccagta agcagtgggt tctctagtta
     2401 gccagagagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     2461 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     2521 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     2581 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     2641 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc
     2701 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     2761 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     2821 gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc
     2881 agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa
     2941 ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt caacgcgtat
     3001 atctggcccg tacatcgcga agcagcgcaa aacgcctaac cctaagcaga ttcttcatgc
     3061 aattgtcggt caagccttgc cttgttgtag cttaaatttt gctcgcgcac tactcagcga
     3121 cctccaacac acaagcaggg agcagatact ggcttaacta tgcggcatca gagcagattg
     3181 tactgagagt gcaccatagg ggatcgggag atctcccgat ccgtcgacgt caggtggcac
     3241 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3301 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3361 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3421 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3481 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3541 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3601 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3661 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3721 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3781 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3841 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3901 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3961 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4021 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4081 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4141 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4201 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4261 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4321 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4381 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4441 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4501 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4561 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4621 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4681 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4741 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4801 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4861 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4921 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4981 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5041 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5101 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5161 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5221 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     5281 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     5341 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     5401 ctagctagag gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc
     5461 acaactagaa tgcactcgac gcgttaagat acattgatga gtttggacaa accacaacta
     5521 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     5581 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     5641 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg
     5701 ctgattatga tcagttatct agagaattga tcccctcaga agaactcgtc aagaaggcga
     5761 tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca
     5821 gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag
     5881 cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc
     5941 atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcatcgcc gtcgggcatg
     6001 cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga
     6061 tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc
     6121 gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca
     6181 gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
     6241 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg
     6301 caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc
     6361 agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg
     6421 aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc
     6481 tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatggc cgatcccata
     6541 ttggctgcag gaattatccc gggttgtggc aagcttatca tcgtgttttt caaaggaaaa
     6601 ccacgtcccc gtggttcggg gggcctagac gtttttttaa cctcgactaa acacatgtaa
     6661 agcatgtgca ccgaggcccc agatcagatc ccatacaatg gggtaccttc tgggcatcct
     6721 tcagcccctt gttgaatacg cttgaggaga gccatttgac tctttccaca actatccaac
     6781 tcacaacgtg gcactggggt tgtgccgcct ttgcaggtgt atcttataca cgtggctttt
     6841 ggccgcagag gcacctgtcg ccaggtgggg ggttccgctg cctgcaaagg gtcgctacag
     6901 acgttgtttg tcttcaagaa gcttccagag gaactgcttc cttcacgaca ttcaacagac
     6961 cttgcattcc tttggcgaga ggggaaagac ccctaggaat gctcgtcaag aagacagggc
     7021 caggtttccg ggccctcaca ttgccaaaag acggcaatat ggtggaaaat aacatataga
     7081 caaacgcaca ccggccttat tccaagcggc ttcggccagt aacgttag
//
