LOCUS       sequence-336179-        7699 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free/mammalian expression vector for Human ACE2 receptor with 
            N-term eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7699)
  TITLE     Alexandrov Lab Plasmids
REFERENCE   2  (bases 1 to 7699)
  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_KA1_Human-ACE2
FEATURES             Location/Qualifiers
     source          1..7699
                     /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"
     CDS             2304..2327
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2334..3044
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             2334..3044
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(2634..2653)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2981..3002
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3045..3068
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4887..4904
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4994..5013)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5059..5114
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5113..5132)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      5202..5249
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5262..5967
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5350..5722
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5379..5967)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5976..6009)
                     /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(6177..6764)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6256..6275)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6777..7568,7569..7637))
                     /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     7400..7419
                     /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 cgcgcgccgg gggggggggg ggggggsggg gggsggggsg gggsgrggcg
     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 tttaaccatg acagtaatgt ataaagtctg taaagacatt aaacacgtaa
     2281 gtgaaaccat gggcacctct ggtcaccacc atcaccacca tcaccacggc acaagcaagg
     2341 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gttggacggc gacgtaaacg
     2401 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     2461 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     2521 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     2581 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     2641 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     2701 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     2761 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     2821 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     2881 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     2941 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     3001 tgaccgccgc cgggatcact ctcggcatgg acgagctata caagctggag gtgctcttcc
     3061 agggtccgag cacaattgag gaacaggcga aaacattcct ggataaattt aatcatgaag
     3121 cagaggactt gttctaccaa tctagcttgg cgtcatggaa ttacaatacc aatatcacgg
     3181 aggaaaacgt gcaaaatatg aataacgcgg gagacaaatg gtcagcgttt ctaaaagaac
     3241 agagtacact tgctcagatg tatccacttc aggagattca aaacttaacg gtgaagctgc
     3301 aactgcaagc tcttcaacaa aacgggagct ctgttctgtc cgaggataag tctaagaggc
     3361 taaacactat tttgaacaca atgtccacta tttattccac aggaaaggtc tgtaaccccg
     3421 acaatcctca agagtgtctg cttctagaac ctggcttgaa cgaaataatg gctaattcac
     3481 tggattataa cgagcgtctt tgggcttggg agtcctggag aagtgaagtt ggtaagcagc
     3541 tacgtccttt atacgaagaa tacgtggtgt taaaaaatga gatggcaaga gccaatcact
     3601 acgaggacta cggcgactat tggcgtggag attacgaggt caacggtgta gatggttatg
     3661 actactccag aggccaatta atagaagatg ttgagcacac gttcgaagaa atcaagccct
     3721 tgtatgaaca cctacacgcc tacgttaggg ccaagttaat gaatgcgtat cctagctata
     3781 taagcccgat aggatgcttg cccgcacact tgcttggcga catgtggggc cgtttctgga
     3841 cgaacctata ctccttaaca gtaccgttcg gtcagaaacc taatattgat gtgacagacg
     3901 caatggtcga tcaggcgtgg gacgcccaaa ggattttcaa ggaagcggag aaattctttg
     3961 tctcagtggg actacctaac atgacacaag gattctggga gaacagcatg ctgactgacc
     4021 caggcaacgt ccagaaggca gtatgtcatc ccacggcgtg ggacttgggt aagggggatt
     4081 ttagaatact aatgtgcacc aaggtgacaa tggacgattt tttgacggca caccacgaga
     4141 tgggtcatat tcagtacgac atggcatacg cggcacaacc gtttttgttg aggaatgggg
     4201 ctaacgaggg ctttcacgaa gccgttggcg agataatgtc tttatctgcg gctacgccta
     4261 agcatttaaa atccatagga ctactgagcc ccgattttca ggaggataat gaaacggaga
     4321 ttaattttct attgaaacaa gctctaacta ttgttggaac gttaccgttt acatatatgc
     4381 tagagaagtg gagatggatg gttttcaagg gcgagatacc taaagaccaa tggatgaaaa
     4441 agtggtggga gatgaagcgt gaaatcgtag gagttgttga gccagtccct cacgatgaga
     4501 cttattgtga ccctgcaagt ttgttccacg tatcaaatga ttattcattc attaggtact
     4561 acacgaggac gctataccag ttccaattcc aagaagcact ttgccaggcg gctaaacatg
     4621 agggaccctt gcacaagtgc gatataagca actcaacaga ggccggacaa aaattattca
     4681 acatgttgcg tctaggtaaa tccgaacctt ggacccttgc gcttgaaaat gtcgtgggcg
     4741 cgaagaacat gaatgtgcgt cctctgctaa actatttcga gccccttttt acctggctta
     4801 aggatcagaa taagaacagt ttcgtcggat ggagtaccga ctggtcccca tacgccgatt
     4861 aagccagctt tctagaccag tttaaacatc accatcacca tcactaagtg attaacctca
     4921 ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata
     4981 ccactgagat cgatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg
     5041 agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt
     5101 ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg
     5161 agtatttggt ttagagtttg gcaacatatg cccatatgta actagcataa ccccttgggg
     5221 cctctaaacg ggtcttgagg ggttttttgc tgaaagcatg cggaggaaat tctccttgaa
     5281 gtttccctgg tgttcaaagt aaaggagttt gcaccagacg cacctctgtt cactggtccg
     5341 gcgtattaaa acacgataca ttgttattag tacatttatt aagcgctaga ttctgtgcgt
     5401 tgttgattta cagacaattg ttgtacgtat tttaataatt cattaaattt ataatcttta
     5461 gggtggtatg ttagagcgaa aatcaaatga ttttcagcgt ctttatatct gaatttaaat
     5521 attaaatcct caatagattt gtaaaatagg tttcgattag tttcaaacaa gggttgtttt
     5581 tccgaaccga tggctggact atctaatgga ttttcgctca acgccacaaa acttgccaaa
     5641 tcttgtagca gcaatctagc tttgtcgata ttcgtttgtg ttttgttttg taataaaggt
     5701 tcgacgtcgt tcaaaatatt atgcgctttt gtatttcttt catcactgtc gttagtgtac
     5761 aattgactcg acgtaaacac gttaaataga gcttggacat atttaacatc gggcgtgtta
     5821 gctttattag gccgattatc gtcgtcgtcc caaccctcgt cgttagaagt tgcttccgaa
     5881 gacgattttg ccatagccac acgacgccta ttaattgtgt cggctaacac gtccgcgatc
     5941 aaatttgtag ttgagctttt tggaattgcg atcgcataac ttcgtatagc atacattata
     6001 cgaagttata agctcggaac gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     6061 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     6121 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     6181 cataggctcc gccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6241 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6301 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6361 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6421 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6481 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6541 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6601 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6661 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6721 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgttac
     6781 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6841 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6901 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6961 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     7021 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     7081 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     7141 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     7201 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7261 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7321 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7381 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7441 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7501 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     7561 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7621 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7681 tgtctcatgt ccgcgcgtt
//
