LOCUS       sequence-335107-        8161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Weak human ACE2 abundance from Bxb1 landing pad.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8161)
  AUTHORS   Shukla N, Roelle SM, Suzart VG, Bruchez AM, Matreyek KA
  TITLE     Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2
            spike mediated infection.
  JOURNAL   PLoS Pathog. 2021 Jul 16;17(7):e1009715. doi: 
            10.1371/journal.ppat.1009715. eCollection 2021 Jul.
  PUBMED    34270613
REFERENCE   2  (bases 1 to 8161)
  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 
            Pathog."; date: "2021-07-16"; volume: "17(7):e1009715. doi"; pages: 
            " 10.1371/journal.ppat.1009715. eCollection 2021 Jul"
COMMENT     Sequence Label: AttB_[Kozak-mut]ACE2_IRES-mCherry-H2A-P2A-PuroR
FEATURES             Location/Qualifiers
     source          1..8161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     123..142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     376..393
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    510..531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(546..551,552..569,570..576)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    584..600
                     /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)."
     primer_bind     589..611
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     608..624
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     608..624
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        645..663
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    672..793
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(683..702)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     737..756
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(984..1004)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             complement(1498..1554)
                     /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     complement(2265..2284)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2534..2552
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_feature    join(2698..2706,2707..2709,2710..3284)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2871..2890)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     3100..3117
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     protein_bind    5884..5921
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     primer_bind     complement(5994..6013)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5995..6013)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6020..6037)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6020..6036)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6029..6051)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6178..6633
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6265..6284)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6475..6496
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6659..6763
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6764..6832,6833..7624)
                     /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     complement(6982..7001)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7795..8161,1..222)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
       61 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      121 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      181 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      241 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      301 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      361 cgaacgaccg agtgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
      421 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      481 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
      541 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
      601 tttcacacag gaaacagcta tgaccatgat tacgccaagc tcgaaattaa ccctcactaa
      661 aggggatctc ctaagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
      721 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
      781 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
      841 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatca
      901 ggatccctat tacttgcggg tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc
      961 ggcggtgacg gtgaagccga gccgctcgta gaaggggagg ttgcggggcg cggaggtctc
     1021 caggaaggcg ggcaccccgg cgcgctcggc cgcctccact ccggggagca cgacggcgct
     1081 gcccagaccc ttgccctggt ggtcgggcga gacgccgacg gtggccagga accacgcggg
     1141 ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga
     1201 accgctcaac tcggccatgc gcgggccgat ctcggcgaac accgcccccg cttcgacgct
     1261 ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc gcgacccaca ccttgccgat
     1321 gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg
     1381 gtccggatcg acggtgtggc gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg
     1441 tacggccctg gggacgtcgt cgcgggtggc gaggcgcacc gtgggcttgt actcggtagg
     1501 tccagggttc tcctccacgt ctccagcctg cttcagcagg ctgaagttag tagctccgct
     1561 gttatctaga tccggtggat cccgtttgcc tttggccttg tggtgactct cggtcttctt
     1621 gggcagtagc acagcctgga tgttgggcag gacaccaccc tgagcgatgg ttactttgcc
     1681 cagcagcttg ttgagctcct cgtcgttgcg gatggccagc tgcaagtggc gcgggataat
     1741 gcgggtcttc ttgttgtcgc gggcggcgtt gcccgccagc tccaggatct cggcagtcag
     1801 gtactccagc accgccgcca ggtacaccgg cgcgccggcc ccgacccgct cagcatagtt
     1861 gcccttgcgg agcagtcggt gcactcggcc cacggggaac tggagaccgg cacgagaaga
     1921 gcgggtcttg gctttggcgc gagctttgcc tccctgcttg ccgcgtcccg acatcagaat
     1981 tcgaagcttg agctcgagat ctgagtagcc gccggtggag tggcggccct cggcgcgttc
     2041 gtactgttcc acgatggtgt agtcctcgtt gtgggaggtg atgtccaact tgatgttgac
     2101 gttgtaggcg ccgggcagct gcacgggctt cttggccttg taggtggtct tgacctcagc
     2161 gtcgtagtgg ccgccgtcct tcagcttcag cctctgcttg atctcgccct tcagggcgcc
     2221 gtcctcgggg tacatccgct cggaggaggc ctcccagccc atggtcttct tctgcattac
     2281 ggggccgtcg gaggggaagt tggtgccgcg cagcttcacc ttgtagatga actcgccgtc
     2341 ctgcagggag gagtcctggg tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg
     2401 ctcccacttg aagccctcgg ggaaggacag cttcaagtag tcggggatgt cggcggggtg
     2461 cttcacgtag gccttggagc cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg
     2521 cagggggcca cccttggtca ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc
     2581 ctcgccctcg ccctcgatct cgaactcgtg gccgttcacg gagccctcca tgtgcacctt
     2641 gaagcgcatg aactccttga tgatggccat gttatcctcc tcgcccttgc tcaccatggt
     2701 tgtggccata ttatcatcgt gtttttcaaa ggaaaaccac gtccccgtgg ttcggggggc
     2761 ctagacgttt ttttaacctc gactaaacac atgtaaagca tgtgcaccga ggccccagat
     2821 cagatcccat acaatggggt accttctggg catccttcag ccccttgttg aatacgcttg
     2881 aggagagcca tttgactctt tccacaacta tccaactcac aacgtggcac tggggttgtg
     2941 ccgcctttgc aggtgtatct tatacacgtg gcttttggcc gcagaggcac ctgtcgccag
     3001 gtggggggtt ccgctgcctg caaagggtcg ctacagacgt tgtttgtctt caagaagctt
     3061 ccagaggaac tgcttccttc acgacattca acagaccttg cattcctttg gcgagagggg
     3121 aaagacccct aggaatgctc gtcaagaaga cagggccagg tttccgggcc ctcacattgc
     3181 caaaagacgg caatatggtg gaaaataaca tatagacaaa cgcacaccgg ccttattcca
     3241 agcggcttcg gccagtaacg ttaggggggg gagagggaga ggggcagtct ctctgattcc
     3301 acagtgatca gttatctaga tccggtggat cccgggcccg cggtaccgtc gactgcagac
     3361 ctgaagagag ggagaaggag gtcagaccgg tgaattctta agttactaag cgggcgccac
     3421 ctgggaggtc tcggtaccaa aggaggtctg aacatcatca gtgttttgga atcctggatt
     3481 attttctcct ttgctaatat cgatggaggc ataaggattt tctccacttc ttgctttatt
     3541 tttcttcttc cgatctctga tcccagtgaa gatcaggatg acaatgccaa ccactatcac
     3601 tcccatcaca actccaaaaa caatcagcca tatggaaaca gggggctggt taggaggtcc
     3661 aagtgttggc tgtatcccca gaaactctag gctgttgtca ttcagacgga aagcatcatt
     3721 gatacggctc cgggacatcc tgatggcctt ttcaacttca gttctaggaa tgatatcaga
     3781 cacattttta ggtgcagtga caaagaaatt aaaggagatt cttggtttca aattagccac
     3841 tcgcacatcc tcctccccaa aaagaatcat ctgatttttt acttttaaaa agtactgcct
     3901 catagcatat gcaacagatg atcggaacag gtacatttca ttgtcgttcc attcatatgc
     3961 tttatctcca agagctgatt ttaggcttat cctcactttg atgctttggt ctgcatatgg
     4021 actccagtcg gtactccatc ccacaaaaga attcttgttc tggtctttca gccaggtaaa
     4081 taagggctca aagtagttga gcagtggcct tacattcatg ttctttgctc ctacaacatt
     4141 ttccaatgct agggtccagg gttctgattt tccaagcctc agcatattga acagtttctg
     4201 tccagcttct gtagagtttg agatgtcaca tttgtgcaga gggccttcat gtttagctgc
     4261 ttgacaaagt gcttcttgaa actggaattg gtaaagggtc cttgtgtaat atcgaatgaa
     4321 tgagtaatca ttagaaacat ggaacagaga tgcggggtca cagtatgttt catcatgggg
     4381 cacaggttcc accaccccaa ctatctctcg cttcatctcc caccactttt tcatccactg
     4441 gtctttggga atttcccctt taaagaccat ccacctccac ttctctaaca tgtaagtaaa
     4501 tggcagagtc ccaacaatcg tgagtgcttg tttgagcagg aagtttattt ctgtttcatt
     4561 gtcttcttga aaatcgggtg acagaagacc aatggatttt aaatgcttag gtgtggctgc
     4621 agaaagtgac atgatttccc caacagcttc atggaatcct tcattagctc catttcttag
     4681 cagaaaaggt tgtgcagcat atgccatatc atactggata tgccccatct catgatgagc
     4741 tgtcaggaag tcgtccattg tcacctttgt gcacataagg atcctgaagt cgcccttccc
     4801 caggtcccaa gctgtgggat ggcagactgc tttctgaaca tttcctgggt ccgttagcat
     4861 ggaattttcc cagaatcctt gagtcatatt aggaagacca acagatacaa agaacttctc
     4921 ggcctccttg aatattctct gtgcatccca ggcctggtcc accattgcat cagtaacatc
     4981 tatgtttggt ttctgtccaa agggaactgt caaagagtac agatttgtcc aaaatctacc
     5041 ccacatatca ccaagcaaat gagcagggag gcatccaatt ggactgatat aggaaggata
     5101 ggcattcatc aactttgccc tcacataggc atgaagatgt tcatataatg gtttaatctc
     5161 ttcaaaggta tgttccacat cttcaatcaa ctggccgcgg ctgtagtcat agccatctac
     5221 cccatttact tcatagtctc ctctccaata atccccatag tcctcataat gatttgctct
     5281 tgccatctca tttttcaaga ccacatactc ttcatataat ggcctcagct gcttgccgac
     5341 ctcagatctc cagctttccc aagcccagag cctctcattg tagtctaaac tgtttgccat
     5401 tatttcattc aaacctggtt caagtaataa gcattcttgt ggattatctg ggttacaaac
     5461 ttttccagta ctgtagatgg tgctcattgt atttagaatt gtgttcaacc gtttgctctt
     5521 gtcttctgag agcactgaag acccattttg ctgaagagcc tgcagctgaa gcttgactgt
     5581 gagattctga atttcttgta gtggatacat ttgggcaagt gtggactgtt cctttaaaaa
     5641 ggcagaccat ttgtccccag cattattcat gttttggaca ttctcttcag taatattggt
     5701 gttataattc caagaagcaa gtgaactttg atagaacagg tcttcggctt cgtggttaaa
     5761 cttgtccaaa aatgtcttgg cctgttcctc aatggtggac tgagcagcag ttacagcaac
     5821 aaggctgaga aggagccagg aggagctgga catacaatgg tgtagttgcg gtataatctg
     5881 cggatgatcc tgacgacgga gaccgccgtc gtcgacaagc cggctcgagc gctgagtccg
     5941 gaatgccgtg acctacatcg tcgacctcga gggggggccc ggtacccaat tcgccctata
     6001 gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     6061 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     6121 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     6181 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     6241 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     6301 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     6361 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     6421 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     6481 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     6541 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     6601 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     6661 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     6721 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     6781 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     6841 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     6901 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     6961 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     7021 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     7081 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     7141 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     7201 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     7261 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     7321 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     7381 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     7441 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     7501 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     7561 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     7621 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     7681 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     7741 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     7801 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7861 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7921 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     7981 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     8041 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     8101 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     8161 c
//
