LOCUS       sequence-222159-        4758 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 4758)
  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 4758)
  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..4758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      73..82
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      77..86
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    121..245
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(270..275,276..293,294..300)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             354..1013
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDRVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(420..439)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1355..1660
                     /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     1539..1558
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1701..1825)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1923..1941)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1924..1941)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1959..1975)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1959..1975)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1972..1994)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1983..1999
                     /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(2007..2013,2014..2031,2032..2037))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2052..2073
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2190..2207)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2361..2949)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2441..2460)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3120..3911,3912..3980))
                     /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     3743..3762
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3981..4085)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4163..4618)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4300..4321)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4512..4531
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(4744..4758,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4758,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggcccgacgt
       61 cgcatgctcc cggccgccat ggcggccgcg ggaattcgat ttagccctag gtacgtaatc
      121 acaagtttgt acaaaaaagc tgaacgagaa acgtaaaatg atataaatat caatatatta
      181 aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca
      241 ctatggcggc cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg
      301 tgtggatttt gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga
      361 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
      421 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
      481 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
      541 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
      601 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
      661 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
      721 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
      781 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
      841 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaggg
      901 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
      961 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg taaacgcgtg
     1021 gatccggctt actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta
     1081 taagaatata tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca
     1141 gcgtattaca gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc
     1201 aatatctccg gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa
     1261 cgctggaaag cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac
     1321 ggctcttttg ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa
     1381 aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg
     1441 gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga
     1501 actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc
     1561 cagtgtgccg gtctccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga
     1621 catcaaaaac gccattaacc tgatgttctg gggaatataa atgtcaggct cccttataca
     1681 cagccagtct gcaggtcgac catagtgact ggatatgttg tgttttacag tattatgtag
     1741 tctgtttttt atgcaaaatc taatttaata tattgatatt tatatcattt tacgtttctc
     1801 gttcagcttt cttgtacaaa gtggtgtacg tattaattaa gcaatcacta gtgaattcgc
     1861 ggccgcctgc aggtcgacca tatgggagag ctcccaacgc gttggatgca tagcttgagt
     1921 attctatagt gtcacctaaa tagcttggcg taatcatggt catagctgtt tcctgtgtga
     1981 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     2041 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     2101 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     2161 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2221 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2281 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2341 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2401 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2461 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2521 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2581 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2641 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2701 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2761 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     2821 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     2881 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     2941 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3001 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3061 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3121 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3181 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3241 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3301 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3361 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3421 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3481 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3541 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3601 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3661 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3721 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3781 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3841 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     3901 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     3961 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4021 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4081 ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt gaaataccgc acagatgcgt
     4141 aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa attcgcgtta
     4201 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     4261 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     4321 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     4381 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     4441 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     4501 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     4561 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     4621 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     4681 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     4741 tttcccagtc acgacgtt
//
