LOCUS       sequence-387239-        5963 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression of SpyTag-ELP-CarHC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5963)
  AUTHORS   Yang Z, Yang Y, Wang M, Wang T, Fok HKF, Jiang B, Xiao W, Kou S, Guo
            Y, Yan Y, Deng X, Zhang W-B, Sun F
  TITLE     Dynamically Tunable, Macroscopic Molecular Networks Enabled by 
            Cellular Synthesis of 4-Arm Star-like Proteins
  JOURNAL   Matter (2020) 2, 233–249
REFERENCE   2  (bases 1 to 5963)
  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: "Matter 
            (2020) 2, 233–249"
COMMENT     Sequence Label: pQE80l AEC
FEATURES             Location/Qualifiers
     source          1..5963
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     40..58
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(82..96,97..102,103..119,120..126)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    102..118
                     /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)."
     protein_bind    134..150
                     /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)."
     RBS             169..180
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             202..219
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             226..264
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     CDS             1390..1428
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     terminator      1492..1586
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1630..2289
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1696..1715)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2354..2440
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2533..3615)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3577..3596
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3616..3693)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(3777..3799)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3885..4025
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3940..3959
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4040..4057)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4211..4799)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4291..4310)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4970..5761,5762..5830))
                     /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     5593..5612
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5831..5935)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
       61 tttcgtcttc accttgagaa atcataaaaa atttatttgc tttgtgagcg gataacaatt
      121 ataatagatt caattgtgag cggataacaa tttcacacag aattcattaa agaggagaaa
      181 ttaactatga aaggcagcag ccatcatcat catcatcacg tcgacgccca tattgtcatg
      241 gttgatgcat acaagccgac gaagctcgac ggccacggcg tgggtgttcc gggcgtcggt
      301 gtgccgggtg tgggtgtgcc gggcgaaggt gtgccgggcg tcggtgtgcc gggtgttggt
      361 gttccgggcg ttggtgtgcc gggcgttggc gtgccgggcg agggtgtgcc gggcgttggt
      421 gttccgggcg tgggtgtgcc gggcgtgggc gtgccgggcg tcggtgttcc gggcgagggt
      481 gtgccgggcg taggtgtgcc gggcgttggt gagctcccag aagatctggg caccggcctg
      541 ctggaagcac tgctgcgcgg tgatctggcg ggcgccgaag ctctgtttcg tcgtggcctg
      601 cgtttctggg gcccggaagg cgttctggag cacctgctgc tgccggtgct gcgtgaagtg
      661 ggcgaagctt ggcaccgtgg tgaaatcggc gttgcagaag aacacctggc gagcaccttc
      721 ctgcgcgcgc gtctgcagga gctgctggac ctggcaggtt tcccgccggg tccgccggtc
      781 ctggtgacta cgccgccggg cgaacgccac gaaatcggtg cgatgctggc ggcgtaccat
      841 ctgcgtcgta agggcgtccc ggcgctgtat ctgggcccgg atactccgct gccggacctg
      901 cgtgcactgg cgcgccgcct gggtgcaggc gcggtcgtgc tgtctgctgt tctgagcgaa
      961 ccgctgcgtg ctctgcctga cggtgccctg aaagatctgg caccgcgtgt tttcctgggc
     1021 ggccagggcg caggcccgga agaggcacgc cgtctgggtg ccgaatacat ggaagacctg
     1081 aaaggcctgg ctgaagcgct gtggctgccg cgcggtccgg aaaaagaagc aatctaaact
     1141 agtgtgccgg gcgtcggcgt gccgggcgta ggtgttccgg gcgagggtgt tccgggcgtt
     1201 ggtgtgccgg gcgtcggcgt gccgggcgtg ggtgttccgg gcgtaggtgt gccgggcgag
     1261 ggtgtgccgg gcgtgggcgt gccgggcgta ggtgttccgg gcgtaggtgt tccgggcgta
     1321 ggtgttccgg gtgaaggcgt gccgggcgtt ggtgtgccgg gtgtgggcgt gccgggcggg
     1381 ctgctcgacg cccatattgt catggttgat gcatacaagc cgacgaagct cgagtggaag
     1441 aaataaggat ccggctgcta acaaagcccg aagcttaatt agctgagctt ggactcctgt
     1501 tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc
     1561 gccgggcgtt ttttattggt gagaatccaa gccagcttgg cgagattttc aggagctaag
     1621 gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat
     1681 cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt
     1741 cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg
     1801 gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaatttcg tatggcaatg
     1861 aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag
     1921 caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta
     1981 cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg
     2041 tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat
     2101 ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat
     2161 acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat
     2221 ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc
     2281 ggggcgtaat ttttttaagg cagttattgg tgcccttaaa cgcctggggt aatgactctc
     2341 tagcttgagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
     2401 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccctcta gattacgtgc
     2461 agtcgatgat aagctgtcaa acatgagaat tgtgcctaat gagtgagcta acttacatta
     2521 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     2581 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     2641 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     2701 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     2761 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     2821 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     2881 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     2941 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     3001 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     3061 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     3121 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     3181 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     3241 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     3301 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     3361 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     3421 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     3481 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     3541 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     3601 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     3661 accgcgaaag gttttgcacc attcgatggt gtcggaattt cgggcagcgt tgggtcctgg
     3721 ccacgggtgc gcatgatcta gagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc
     3781 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     3841 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag
     3901 tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac
     3961 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     4021 tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4081 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4141 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4201 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4261 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4321 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4381 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4441 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4501 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4561 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4621 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     4681 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4741 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4801 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4861 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4921 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4981 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5041 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5101 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5161 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5221 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5281 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5341 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5401 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5461 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5521 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5581 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5641 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5701 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5761 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5821 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5881 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5941 ttccccgaaa agtgccacct gac
//
