LOCUS       sequence-372760-        8416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express human ACE2 with selectable markers iRFP670 and puromycin 
            resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8416)
  AUTHORS   Roelle SM, Shukla N, Pham AT, Bruchez AM, Matreyek KA
  TITLE     Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor
            binding domains.
  JOURNAL   PLoS Biol. 2022 Jul 27;20(7):e3001738. doi: 
            10.1371/journal.pbio.3001738.
  PUBMED    35895696
REFERENCE   2  (bases 1 to 8416)
  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: "PLoS 
            Biol."; date: "2022-07-27"; volume: "20(7):e3001738. doi"; pages: " 
            10.1371/journal.pbio.3001738"
COMMENT     Sequence Label: AttB_ACE2_IRES_iRFP670-H2A-P2A-PuroR
FEATURES             Location/Qualifiers
     source          1..8416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     65..87
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     79..96
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     80..96
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     103..122
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        103..121
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    195..232
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_feature    join(2832..3406,3407..3409,3410..3418)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2999..3016)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3226..3245
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3419..4351
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="MARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAG
                     AFFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLH
                     RHDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVM
                     LYRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSR
                     IVPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYE
                     PRAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     CDS             4817..4873
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     5367..5387
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5578..5699
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5615..5634)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5669..5688
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(5708..5726)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5747..5763)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5747..5763)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5760..5782)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5771..5787
                     /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(5795..5801,5802..5819,5820..5825))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5840..5861
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5978..5995)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6149..6737)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6229..6248)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6908..7699,7700..7768))
                     /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     7531..7550
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7769..7873)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7899..8354)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8036..8057)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8248..8267
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
       61 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag
      121 ggcgaattgg gtaccgggcc ccccctcgag gtcgacgatg taggtcacgg cattccggac
      181 tcagcgctcg agccggcttg tcgacgacgg cggtctccgt cgtcaggatc atccgcagat
      241 tataccgcaa ctacacgcca ccatgtccag ctcctcctgg ctccttctca gccttgttgc
      301 tgtaactgct gctcagtcca ccattgagga acaggccaag acatttttgg acaagtttaa
      361 ccacgaagcc gaagacctgt tctatcaaag ttcacttgct tcttggaatt ataacaccaa
      421 tattactgaa gagaatgtcc aaaacatgaa taatgctggg gacaaatggt ctgccttttt
      481 aaaggaacag tccacacttg cccaaatgta tccactacaa gaaattcaga atctcacagt
      541 caagcttcag ctgcaggctc ttcagcaaaa tgggtcttca gtgctctcag aagacaagag
      601 caaacggttg aacacaattc taaatacaat gagcaccatc tacagtactg gaaaagtttg
      661 taacccagat aatccacaag aatgcttatt acttgaacca ggtttgaatg aaataatggc
      721 aaacagttta gactacaatg agaggctctg ggcttgggaa agctggagat ctgaggtcgg
      781 caagcagctg aggccattat atgaagagta tgtggtcttg aaaaatgaga tggcaagagc
      841 aaatcattat gaggactatg gggattattg gagaggagac tatgaagtaa atggggtaga
      901 tggctatgac tacagccgcg gccagttgat tgaagatgtg gaacatacct ttgaagagat
      961 taaaccatta tatgaacatc ttcatgccta tgtgagggca aagttgatga atgcctatcc
     1021 ttcctatatc agtccaattg gatgcctccc tgctcatttg cttggtgata tgtggggtag
     1081 attttggaca aatctgtact ctttgacagt tccctttgga cagaaaccaa acatagatgt
     1141 tactgatgca atggtggacc aggcctggga tgcacagaga atattcaagg aggccgagaa
     1201 gttctttgta tctgttggtc ttcctaatat gactcaagga ttctgggaaa attccatgct
     1261 aacggaccca ggaaatgttc agaaagcagt ctgccatccc acagcttggg acctggggaa
     1321 gggcgacttc aggatcctta tgtgcacaaa ggtgacaatg gacgacttcc tgacagctca
     1381 tcatgagatg gggcatatcc agtatgatat ggcatatgct gcacaacctt ttctgctaag
     1441 aaatggagct aatgaaggat tccatgaagc tgttggggaa atcatgtcac tttctgcagc
     1501 cacacctaag catttaaaat ccattggtct tctgtcaccc gattttcaag aagacaatga
     1561 aacagaaata aacttcctgc tcaaacaagc actcacgatt gttgggactc tgccatttac
     1621 ttacatgtta gagaagtgga ggtggatggt ctttaaaggg gaaattccca aagaccagtg
     1681 gatgaaaaag tggtgggaga tgaagcgaga gatagttggg gtggtggaac ctgtgcccca
     1741 tgatgaaaca tactgtgacc ccgcatctct gttccatgtt tctaatgatt actcattcat
     1801 tcgatattac acaaggaccc tttaccaatt ccagtttcaa gaagcacttt gtcaagcagc
     1861 taaacatgaa ggccctctgc acaaatgtga catctcaaac tctacagaag ctggacagaa
     1921 actgttcaat atgctgaggc ttggaaaatc agaaccctgg accctagcat tggaaaatgt
     1981 tgtaggagca aagaacatga atgtaaggcc actgctcaac tactttgagc ccttatttac
     2041 ctggctgaaa gaccagaaca agaattcttt tgtgggatgg agtaccgact ggagtccata
     2101 tgcagaccaa agcatcaaag tgaggataag cctaaaatca gctcttggag ataaagcata
     2161 tgaatggaac gacaatgaaa tgtacctgtt ccgatcatct gttgcatatg ctatgaggca
     2221 gtacttttta aaagtaaaaa atcagatgat tctttttggg gaggaggatg tgcgagtggc
     2281 taatttgaaa ccaagaatct cctttaattt ctttgtcact gcacctaaaa atgtgtctga
     2341 tatcattcct agaactgaag ttgaaaaggc catcaggatg tcccggagcc gtatcaatga
     2401 tgctttccgt ctgaatgaca acagcctaga gtttctgggg atacagccaa cacttggacc
     2461 tcctaaccag ccccctgttt ccatatggct gattgttttt ggagttgtga tgggagtgat
     2521 agtggttggc attgtcatcc tgatcttcac tgggatcaga gatcggaaga agaaaaataa
     2581 agcaagaagt ggagaaaatc cttatgcctc catcgatatt agcaaaggag aaaataatcc
     2641 aggattccaa aacactgatg atgttcagac ctcctttggt accgagacct cccaggtggc
     2701 gcccgcttag taacttaaga attcaccggt ctgacctcct tctccctctc ttcaggtctg
     2761 cagtcgacgg taccgcgggc ccgggatcca ccggatctag ataactgatc actgtggaat
     2821 cagagagact gcccctctcc ctctcccccc cctaacgtta ctggccgaag ccgcttggaa
     2881 taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat
     2941 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct
     3001 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct
     3061 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc
     3121 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa
     3181 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc
     3241 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg
     3301 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc
     3361 ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaaccat
     3421 ggcgcgtaag gtcgatctca cctcctgcga tcgcgagccg atccacatcc ccggcagcat
     3481 tcagccgtgc ggctgcctgc tagcctgcga cgcgcaggcg gtgcggatca cgcgcattac
     3541 ggaaaatgcc ggcgcgttct ttggacgcga aactccgcgg gtcggtgagc tactcgccga
     3601 ttacttcggc gagaccgaag cccatgcgct gcgcaacgca ctggcgcagt cctccgatcc
     3661 aaagcgaccg gcgctgatct tcggttggcg cgacggcctg accggccgca ccttcgacat
     3721 ctcactgcat cgccatgacg gtacatcgat catcgagttc gagcctgcgg cggccgaaca
     3781 ggccgacaat ccgctgcggc tgacgcggca gatcatcgcg cgcaccaaag aactgaagtc
     3841 gctcgaagag atggccgcac gggtgccgcg ctatctgcag gcgatgctcg gctatcaccg
     3901 cgtgatgttg taccgcttcg cggacgacgg ctccgggatg gtgatcggcg aggcgaagcg
     3961 cagcgacctc gagagctttc tcggtcagca ctttccggcg tcgctggtcc cgcagcaggc
     4021 gcggctactg tacttgaaga acgcgatccg cgtggtctcg gattcgcgcg gcatcagcag
     4081 ccggatcgtg cccgagcacg acgcctccgg cgccgcgctc gatctgtcgt tcgcgcacct
     4141 gcgcagcatc tcgccctgcc atctcgaatt tctgcggaac atgggcgtca gcgcctcgat
     4201 gtcgctgtcg atcatcattg acggcacgct atggggattg atcatctgtc atcattacga
     4261 gccgcgtgcc gtgccgatgg cgcagcgcgt cgcggccgaa atgttcgccg acttcttatc
     4321 gctgcacttc accgccgccc accaccaacg ctccaccggc ggctactcag atctcgagct
     4381 caagcttcga attctgatgt cgggacgcgg caagcaggga ggcaaagctc gcgccaaagc
     4441 caagacccgc tcttctcgtg ccggtctcca gttccccgtg ggccgagtgc accgactgct
     4501 ccgcaagggc aactatgctg agcgggtcgg ggccggcgcg ccggtgtacc tggcggcggt
     4561 gctggagtac ctgactgccg agatcctgga gctggcgggc aacgccgccc gcgacaacaa
     4621 gaagacccgc attatcccgc gccacttgca gctggccatc cgcaacgacg aggagctcaa
     4681 caagctgctg ggcaaagtaa ccatcgctca gggtggtgtc ctgcccaaca tccaggctgt
     4741 gctactgccc aagaagaccg agagtcacca caaggccaaa ggcaaacggg atccaccgga
     4801 tctagataac agcggagcta ctaacttcag cctgctgaag caggctggag acgtggagga
     4861 gaaccctgga cctaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     4921 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     4981 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     5041 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     5101 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     5161 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     5221 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
     5281 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     5341 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     5401 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagta
     5461 atagggatcc tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     5521 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     5581 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     5641 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag
     5701 gagatcccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc
     5761 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5821 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5881 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5941 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcact
     6001 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6061 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6121 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6181 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6241 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6301 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6361 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6421 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6481 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6541 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6601 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6661 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6721 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6781 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6841 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6901 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6961 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     7021 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     7081 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7141 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7201 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7261 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7321 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7381 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7441 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7501 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7561 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7621 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7681 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7741 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7801 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7861 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     7921 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     7981 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     8041 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     8101 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     8161 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     8221 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     8281 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     8341 gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     8401 gcgggcctct tcgcta
//
