LOCUS       sequence-336170-        7778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for cell-free and mamalian expression 
            with C-term mCherry, 8xHis and Myc tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7778)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 7778)
  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"
COMMENT     Sequence Label: pmCellFree_KA3_Cterm-mCherry
FEATURES             Location/Qualifiers
     source          1..7778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1140..1443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1449..1726
                     /label=chicken beta-actin promoter
     primer_bind     2005..2027
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2089..2108
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2147..2166
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2147..2165
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2300..2424
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(2471..2776)
                     /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     complement(2573..2592)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3118..3777)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3692..3711
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3778..3880)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(4027..4151)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             4153..4176
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(4313..4331)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4581..4600
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4879..4902
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             4903..4932
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4966..4983
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5073..5092)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5138..5193
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5192..5211)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      5281..5328
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5341..6046
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5429..5801
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5458..6046)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(6055..6088)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(6256..6843)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6335..6354)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6856..7647,7648..7716))
                     /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     7479..7498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggccg
     1021 taatgagacg cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctct
     1081 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     1141 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     1201 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     1261 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     1321 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     1381 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     1441 atgcatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     1501 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     1561 gggggggggg cgcgcgccrg ggggggsggg gsggggsgrg gggsggggsg gggcgaggcg
     1621 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
     1681 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1741 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1801 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctccttcgg gctgtaatta
     1861 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
     1921 ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc
     1981 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
     2041 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     2101 ttattgtgct gtctcatcat tttggcaaag aattggatcg gacgtctaat acgactcact
     2161 atagggacat cttaagttta ttttatttta ttttatttta ttttatttta ttttatttta
     2221 ttttatttta ttttatttaa ccatgacagt aatgtataaa gtctgtaaag acattaaaca
     2281 cgtaagtgaa accatgggca caagtttgta caaaaaagct gaacgagaaa cgtaaaatga
     2341 tataaatatc aatatattaa attagatttt gcataaaaaa cagactacat aatactgtaa
     2401 aacacaacat atccagtcac tatggggccc gtcgacctgc agactggctg tgtataaggg
     2461 agcctgacat ttatattccc cagaacatca ggttaatggc gtttttgatg tcattttcgc
     2521 ggtggctgag atcagccact tcttccccga taacggagac cggcacactg gccatatcgg
     2581 tggtcatcat gcgccagctt tcatccccga tatgcaccac cgggtaaagt tcacgggaga
     2641 ctttatctga cagcagacgt gcactggcca gggggatcac catccgtcgc ccgggcgtgt
     2701 caataatatc actctgtaca tccacaaaca gacgataacg gctctctctt ttataggtgt
     2761 aaaccttaaa ctgcatttca ccagcccctg ttctcgtcag caaaagagcc gttcatttca
     2821 ataaaccggg cgacctcagc catcccttcc tgattttccg ctttccagcg ttcggcacgc
     2881 agacgacggg cttcattctg catggttgtg cttaccagac cggagatatt gacatcatat
     2941 atgccttgag caactgatag ctgtcgctgt caactgtcac tgtaatacgc tgcttcatag
     3001 catacctctt tttgacatac ttcgggtata catatcagta tatattctta taccgcaaaa
     3061 atcagcgcgc aaatacgcat actgttatct ggcttttagt aagccggatc cacgcgttta
     3121 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg
     3181 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc
     3241 ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac
     3301 gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc
     3361 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata
     3421 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc
     3481 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc
     3541 gtctttcatt gccatacgga attccggatg agcattcatc aggcgggcaa gaatgtgaat
     3601 aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc
     3661 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc
     3721 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt
     3781 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat
     3841 ttcattatgg tgaaagttgg aacctcttgc gtgccgatca acgtctcatt ttcgccaaaa
     3901 gttggcccag ggcttcccgg tatcaacagg gacaccagga tttatttatt ctgcgaagtg
     3961 atcttccgtc acaggtattt attcggcgca aagtgcgtcg ggtgatgctg ccaacttagc
     4021 aagcttcata gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc
     4081 aaaatctaat ttaatatatt gatatttata tcattttacg tttctcgttc agctttcttg
     4141 tacaaagtgg tgctggaggt gctcttccag ggcccgaagg gcgaggagga taacatggcc
     4201 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     4261 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     4321 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     4381 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     4441 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     4501 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     4561 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ctatgggctg ggaggcctcc
     4621 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     4681 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     4741 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     4801 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     4861 gacgagctgt acaagaccca ccaccatcac caccatcacc acgagcagaa gctgatctcg
     4921 gaggaggatc tgtaagcggc cgccagcttt ctagaccagt ttaaacatca ccatcaccat
     4981 cactaagtga ttaacctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca
     5041 atgccctggc tcacaaatac cactgagatc gatctttttc cctctgccaa aaattatggg
     5101 gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt
     5161 gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat
     5221 catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgtaa
     5281 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaagcatgc
     5341 ggaggaaatt ctccttgaag tttccctggt gttcaaagta aaggagtttg caccagacgc
     5401 acctctgttc actggtccgg cgtattaaaa cacgatacat tgttattagt acatttatta
     5461 agcgctagat tctgtgcgtt gttgatttac agacaattgt tgtacgtatt ttaataattc
     5521 attaaattta taatctttag ggtggtatgt tagagcgaaa atcaaatgat tttcagcgtc
     5581 tttatatctg aatttaaata ttaaatcctc aatagatttg taaaataggt ttcgattagt
     5641 ttcaaacaag ggttgttttt ccgaaccgat ggctggacta tctaatggat tttcgctcaa
     5701 cgccacaaaa cttgccaaat cttgtagcag caatctagct ttgtcgatat tcgtttgtgt
     5761 tttgttttgt aataaaggtt cgacgtcgtt caaaatatta tgcgcttttg tatttctttc
     5821 atcactgtcg ttagtgtaca attgactcga cgtaaacacg ttaaatagag cttggacata
     5881 tttaacatcg ggcgtgttag ctttattagg ccgattatcg tcgtcgtccc aaccctcgtc
     5941 gttagaagtt gcttccgaag acgattttgc catagccaca cgacgcctat taattgtgtc
     6001 ggctaacacg tccgcgatca aatttgtagt tgagcttttt ggaattgcga tcgcataact
     6061 tcgtatagca tacattatac gaagttataa gctcggaacg ctgcgctcgg tcgttcggct
     6121 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     6181 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     6241 cgcgttgctg gcgtttttcc ataggctccg ccccctgacg agcatcacaa aaatcgacgc
     6301 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     6361 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     6421 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     6481 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6541 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6601 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6661 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6721 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6781 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6841 caagaagatc ctttgttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6901 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6961 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     7021 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     7081 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     7141 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     7201 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     7261 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7321 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7381 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7441 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7501 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7561 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7621 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7681 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7741 aagcatttat cagggttatt gtctcatgtc cgcgcgtt
//
