LOCUS       sequence-214661-        4751 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector only containing the Gateway cassette and 
            pGEM-T Easy backbone. This can be used with Gateway-compatible 
            Homology-Directed Repair..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4751)
  AUTHORS   Mallett DR, Chang M, Cheng X, Bezanilla M
  TITLE     Efficient and modular CRISPR-Cas9 vector system for Physcomitrella 
            patens.
  JOURNAL   Plant Direct. 2019 Sep 12;3(9):e00168. doi: 10.1002/pld3.168. 
            eCollection 2019 Sep.
  PUBMED    31523744
REFERENCE   2  (bases 1 to 4751)
  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: "Plant 
            Direct."; date: "2019-09-12"; volume: "3(9):e00168. doi"; pages: " 
            10.1002/pld3.168. eCollection 2019 Sep"
COMMENT     Sequence Label: pGEM-gate
FEATURES             Location/Qualifiers
     source          1..4751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      37..46
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    77..201
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(226..231,232..249,250..256)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             310..969
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(376..395)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1311..1616
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1495..1514
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1657..1781)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1876..1894)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1877..1894)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1912..1928)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1912..1928)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1925..1947)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1936..1952
                     /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(1960..1966,1967..1984,1985..1990))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2005..2026
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2143..2160)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2314..2902)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2394..2413)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3073..3864,3865..3933))
                     /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     3696..3715
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3934..4038)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4116..4571)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4253..4274)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4465..4484
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4697..4719
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4711..4728
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4712..4728
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(4735..4751,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4735..4751,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat
       61 tcctaggtac gtaatcacaa gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat
      121 aaatatcaat atattaaatt agattttgca taaaaaacag actacataat actgtaaaac
      181 acaacatatc cagtcactat ggcggccgca ttaggcaccc caggctttac actttatgct
      241 tccggctcgt ataatgtgtg gattttgagt taggatccgt cgagattttc aggagctaag
      301 gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat
      361 cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt
      421 cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg
      481 gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg
      541 aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag
      601 caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta
      661 cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg
      721 tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat
      781 ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat
      841 acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat
      901 ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc
      961 ggggcgtaaa cgcgtggatc cggcttacta aaagccagat aacagtatgc gtatttgcgc
     1021 gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat gtcaaaaaga
     1081 ggtgtgctat gaagcagcgt attacagtga cagttgacag cgacagctat cagttgctca
     1141 aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga atgaagcccg
     1201 tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg
     1261 gtttattgaa atgaacggct cttttgctga cgagaacagg gactggtgaa atgcagttta
     1321 aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata
     1381 ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag
     1441 ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga
     1501 tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca
     1561 gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt
     1621 caggctccct tatacacagc cagtctgcag gtcgaccata gtgactggat atgttgtgtt
     1681 ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata
     1741 tcattttacg tttctcgttc agctttcttg tacaaagtgg tgtacgtatt aattaaatca
     1801 ctagtgaatt cgcggccgcc tgcaggtcga ccatatggga gagctcccaa cgcgttggat
     1861 gcatagcttg agtattctat agtgtcacct aaatagcttg gcgtaatcat ggtcatagct
     1921 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     1981 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     2041 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2101 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2161 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2221 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2281 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2341 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2401 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2461 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2521 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2581 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2641 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2701 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2761 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2821 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2881 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2941 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3001 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3061 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3121 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3181 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3241 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3301 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3361 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3421 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3481 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3541 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3601 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3661 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3721 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3781 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3841 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     3901 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     3961 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4021 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgatgcgg tgtgaaatac
     4081 cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt
     4141 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     4201 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     4261 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     4321 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     4381 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     4441 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     4501 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     4561 acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     4621 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     4681 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac
     4741 gactcactat a
//
