LOCUS       sequence-394529-        7910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre reporter for mammalian cells with Gap43 membrane anchor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7910)
  AUTHORS   Strecker J, Demircioglu FE, Li D, Faure G, Wilkinson ME, Gootenberg 
            JS, Abudayyeh OO, Nishimasu H, Macrae RK, Zhang F
  TITLE     RNA-activated protein cleavage with a CRISPR-associated 
            endopeptidase.
  JOURNAL   Science. 2022 Nov 25;378(6622):874-881. doi: 
            10.1126/science.add7450. Epub 2022 Nov 3.
  PUBMED    36423276
REFERENCE   2  (bases 1 to 7910)
  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"; doi: 
            "10.1126/science.add7450"; journalName: "Science"; date: 
            "2022-11-25- 25"; volume: "378"; issue: "6622"; pages: "874-881"
COMMENT     Sequence Label: pCDNA3_Gap43_Csx30_Cre
FEATURES             Location/Qualifiers
     source          1..7910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        170..418
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     170..190
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     341..360
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        538..917
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        918..1121
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1072..1092
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1166..1185
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1166..1184
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1298..1307
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2258..2338
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             2342..2362
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2402..3430
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     primer_bind     complement(3501..3518)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3507..3731
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3777..4205
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3864..3883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4074..4095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4214..4234)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4219..4548
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4399..4534
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4461..4480
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4615..5409
                     /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(4669..4688)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5279..5298
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5583..5704
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5620..5639)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5674..5693
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5753..5769)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5753..5769)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5766..5788)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5777..5793
                     /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(5801..5807,5808..5825,5826..5831))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5846..5867
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5984..6001)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6155..6743)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6235..6254)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6914..7705,7706..7774))
                     /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     7537..7556
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7775..7879)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga cttaattaag agggcctatt
      181 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
      241 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
      301 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
      361 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgt
      421 aatttctact cttgtagatg gagacgatat atcgtctctt tttcaattgc atgaagaatc
      481 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      541 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      601 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      661 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      721 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      781 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      841 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      901 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      961 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     1021 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     1081 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac
     1141 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct
     1201 agcgtttaaa cttaagcttg ccaccatgct gtgctgtatg cggagaacca agcaggtgga
     1261 aaagaacgac gaggatcaga aaatcggcgg agggtccgcc accatgggct ataatcaccc
     1321 gtatccggct gatgtgggta tgcgtattca tcaggttgcc cgcgcggttt tttcgcaaac
     1381 gaacctgtcc ccggcggaac gcctggctgt agccatcgct ggagcgtgct tcaccccgga
     1441 aatcagcgag gaccgtcgtt tggagatcct cttggactgt gaagaacgtg tctgtgaaat
     1501 tgaagcaccg actggtgacg acacgagcgt acgtgttatc aaggatctga aggccctggc
     1561 tgatcatcgt gtccgccacg aaatcccagc cgagtccctc gtgagcctgt ggtttgaaca
     1621 gattgaagcc gcaggcacgg acttcgatac caaaacccct atggatgagc ttgtgctgcg
     1681 tatgctgtcc gataacgtga tcaccctgtc ggtggatcgt aaagccgcga gccaaaccga
     1741 gacagacgac gttaagccgc agaaaggtaa aatcatcccg ttcccggttc cggatatcgc
     1801 gaacgatgag gtggagtacc agaaagctgt gggtatgaaa aaggacaaaa aggccgcgaa
     1861 cgactccaaa gtgaagtttc cgggtctgtt ggagatccaa ggttgccgtg atggcgataa
     1921 ggcgattcta ttggaggaca ctgacgacgc agccgcgaac caccgcaaac tatttagtat
     1981 tctgaaggcg ggtaagctga acagcgcgtt cttcatccaa tctgacgacg gcgagtgggt
     2041 tgaatctgag agtaagccga ccatggaaga caatcgtatt attttgcacg atagccacca
     2101 cagcagcttc gtttggattc tcgacacggg tagcatgcag ctgcgccaaa gcgttaagtg
     2161 cgttaaggat gcgctgaata aaaagacggg ctctgcgaaa aaattgaagc caaaaaccat
     2221 gatcgtgtgg gtcaccattc cgcaagaagg cggatcttac ccatacgatg ttccagatta
     2281 cgcttatccc tacgacgtgc ctgattatgc atacccatat gatgtccccg actatgccgc
     2341 gccgaaaaaa aaacgcaaag tgaacggcgg agggtccgga ggaggcggta gcggaggcgc
     2401 gagcaattta ctgaccgtac accaaaattt gcctgcatta ccggtcgatg caacgagtga
     2461 tgaggttcgc aagaacctga tggacatgtt cagggatcgc caggcgtttt ctgagcatac
     2521 ctggaaaatg cttctgtccg tttgccggtc gtgggcggca tggtgcaagt tgaataaccg
     2581 gaaatggttt cccgcagaac ctgaagatgt tcgcgattat cttctatatc ttcaggcgcg
     2641 cggtctggca gtaaaaacta tccagcaaca tttgggccag ctaaacatgc ttcatcgtcg
     2701 gtccgggctg ccacgaccaa gtgacagcaa tgctgtttca ctggttatgc ggcggatccg
     2761 aaaagaaaac gttgatgccg gtgaacgtgc aaaacaggct ctagcgttcg aacgcactga
     2821 tttcgaccag gttcgttcac tcatggaaaa tagcgatcgc tgccaggata tacgtaatct
     2881 ggcatttctg gggattgctt ataacaccct gttacgtata gccgaaattg ccaggatcag
     2941 ggttaaagat atctcacgta ctgacggtgg gagaatgtta atccatattg gcagaacgaa
     3001 aacgctggtt agcaccgcag gtgtagagaa ggcacttagc ctgggggtaa ctaaactggt
     3061 cgagcgatgg atttccgtct ctggtgtagc tgatgatccg aataactacc tgttttgccg
     3121 ggtcagaaaa aatggtgttg ccgcgccatc tgccaccagc cagctatcaa ctcgcgccct
     3181 ggaagggatt tttgaagcaa ctcatcgatt gatttacggc gctaaggatg actctggtca
     3241 gagatacctg gcctggtctg gacacagtgc ccgtgtcgga gccgcgcgag atatggcccg
     3301 cgctggagtt tcaataccgg agatcatgca agctggtggc tggaccaatg taaatattgt
     3361 catgaactat atccgtaacc tggatagtga aacaggggca atggtgcgcc tgctggaaga
     3421 tggcgactaa gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc
     3481 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3541 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3601 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3661 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3721 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3781 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3841 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3901 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3961 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     4021 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4081 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4141 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4201 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4261 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4321 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4381 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4441 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     4501 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     4561 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4621 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4681 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4741 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     4801 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4861 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4921 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4981 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     5041 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     5101 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     5161 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     5221 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     5281 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     5341 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     5401 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     5461 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5521 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5581 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5641 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5701 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5761 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5821 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5881 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5941 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     6001 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6061 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6121 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6181 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6241 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6301 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6361 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6421 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6481 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6541 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6601 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6661 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6721 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6781 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6841 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6901 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6961 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     7021 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     7081 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     7141 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7201 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7261 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7321 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7381 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7441 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7501 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7561 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7621 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7681 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7741 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7801 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7861 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
