LOCUS       sequence-387236-        5645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression of SpyTag-ELP-CarHCN-ELP-SpyTag, with GRGD site 
            linked with (GGGGS)2 before CarHCN and after first ELP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5645)
  TITLE     Sun lab protein expression plasmids
REFERENCE   2  (bases 1 to 5645)
  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: pQE80l A-CN-A
FEATURES             Location/Qualifiers
     source          1..5645
                     /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             1072..1110
                     /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      1174..1268
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1312..1971
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1378..1397)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2036..2122
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2215..3297)
                     /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     3259..3278
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3298..3375)
                     /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(3459..3481)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3567..3707
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3622..3641
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3722..3739)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3893..4481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3973..3992)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4652..5443,5444..5512))
                     /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     5275..5294
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5513..5617)
                     /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 gagctcggcc gtggtgatgg tggtgggggt
      541 agcggtggtg ggggtagtcc agaagatctg ggcaccggcc tgctggaagc actgctgcgc
      601 ggtgatctgg cgggcgccga agctctgttt cgtcgtggcc tgcgtttctg gggcccggaa
      661 ggcgttctgg agcacctgct gctgccggtg ctgcgtgaag tgggcgaagc ttggcaccgt
      721 ggtgaaatcg gcgttgcaga agaacacctg gcgagcacct tcctgcgcgc gcgtctgcag
      781 gagctgctgg acctggcagg tttcccgccg ggtccgccga ctagtgtgcc gggcgtcggc
      841 gtgccgggcg taggtgttcc gggcgagggt gttccgggcg ttggtgtgcc gggcgtcggc
      901 gtgccgggcg tgggtgttcc gggcgtaggt gtgccgggcg agggtgtgcc gggcgtgggc
      961 gtgccgggcg taggtgttcc gggcgtaggt gttccgggcg taggtgttcc gggtgaaggc
     1021 gtgccgggcg ttggtgtgcc gggtgtgggc gtgccgggcg ggctgctcga cgcccatatt
     1081 gtcatggttg atgcatacaa gccgacgaag ctcgagtgga agaaataagg atccggctgc
     1141 taacaaagcc cgaagcttaa ttagctgagc ttggactcct gttgatagat ccagtaatga
     1201 cctcagaact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg
     1261 gtgagaatcc aagccagctt ggcgagattt tcaggagcta aggaagctaa aatggagaaa
     1321 aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag
     1381 gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc
     1441 tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt
     1501 gcccgcctga tgaatgctca tccggaattt cgtatggcaa tgaaagacgg tgagctggtg
     1561 atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca
     1621 tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat
     1681 gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt
     1741 ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg
     1801 gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg
     1861 ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga
     1921 atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta atttttttaa
     1981 ggcagttatt ggtgccctta aacgcctggg gtaatgactc tctagcttga ggcatcaaat
     2041 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     2101 cgctctcctg agtaggacaa atccgccctc tagattacgt gcagtcgatg ataagctgtc
     2161 aaacatgaga attgtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     2221 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     2281 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     2341 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     2401 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     2461 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     2521 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     2581 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     2641 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     2701 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     2761 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     2821 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     2881 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     2941 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     3001 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     3061 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     3121 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     3181 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     3241 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     3301 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca
     3361 ccattcgatg gtgtcggaat ttcgggcagc gttgggtcct ggccacgggt gcgcatgatc
     3421 tagagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     3481 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     3541 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     3601 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     3661 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     3721 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3781 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3841 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3901 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3961 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4021 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4081 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4141 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4201 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4261 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4321 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4381 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4441 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4501 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4561 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4621 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4681 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4741 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4801 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4861 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4921 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4981 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5041 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5101 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5161 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5221 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5281 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5341 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5401 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5461 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5521 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5581 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5641 ctgac
//
