LOCUS       sequence-319428-        5401 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AGSC domain from Pontibacillus A/G cyclase enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5401)
  TITLE     Bacterial expression plasmids for production of recombinant AGSC 
            domains
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5401)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pGEX4-4T-1 Pontibacillus AGSC
FEATURES             Location/Qualifiers
     source          1..5401
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(180..185,186..201,202..208)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    216..232
                     /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)."
     CDS             255..908
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     866..888
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             915..932
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             933..953
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1368..1385
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1448..1470)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1615..1633)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1701..1805
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1806..1874,1875..2666)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2024..2043)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2837..3425
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3326..3345
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3579..3596
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3669..3746
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3747..4829
                     /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     complement(3766..3785)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4878..4883,4884..4901,4902..4908)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4916..4932
                     /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     4940..4956
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4940..4956
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4952..5401,1..78)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4972..4989)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4972..4988)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4981..5003)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta
       61 tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct
      121 ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt
      181 tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac aatttcacac
      241 aggaaacagt attcatgtcc cctatactag gttattggaa aattaagggc cttgtgcaac
      301 ccactcgact tcttttggaa tatcttgaag aaaaatatga agagcatttg tatgagcgcg
      361 atgaaggtga taaatggcga aacaaaaagt ttgaattggg tttggagttt cccaatcttc
      421 cttattatat tgatggtgat gttaaattaa cacagtctat ggccatcata cgttatatag
      481 ctgacaagca caacatgttg ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg
      541 aaggagcggt tttggatatt agatacggtg tttcgagaat tgcatatagt aaagactttg
      601 aaactctcaa agttgatttt cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc
      661 gtttatgtca taaaacatat ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt
      721 atgacgctct tgatgttgtt ttatacatgg acccaatgtg cctggatgcg ttcccaaaat
      781 tagtttgttt taaaaaacgt attgaagcta tcccacaaat tgataagtac ttgaaatcca
      841 gcaagtatat agcatggcct ttgcagggct ggcaagccac gtttggtggt ggcgaccatc
      901 ctccaaaatc ggatctggtt ccgcgtggat ccgaaaacct ctacttccaa ggttttgact
      961 gggagaagta cttgaatagt ttctcatttc tcccaaagac caaagatttc aacaattata
     1021 taagcccgac agactttgaa gtggtctgtt atgtcaaaaa gaacgactca gaccaataca
     1081 ctcggtactt ccagtcctct tccagtcttg ataagggcat gaaggttatg tttgagcttt
     1141 tcaattatgc caatatgccg attggcaaca taaaatggcg cgtcgttaat tatggcgatg
     1201 aggccgagca atctgagaag gacctcgaat actatatgaa agattttgaa ggcgagtact
     1261 tctgtgagca aagtaccgca tacacagggc ttcattttat ggaatgctac atctataatc
     1321 cggaatccaa gctgatcgca aaagataggt cagggatcta cattaatcat catcatcacc
     1381 atcattagcg gccgcatcgt gactgactga cgatctgcct cgcgcgtttc ggtgatgacg
     1441 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     1501 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     1561 ccatgaccca gtcacgtagc gatagcggag tgtataattc ttgaagacga aagggcctcg
     1621 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     1681 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1741 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     1801 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     1861 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     1921 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     1981 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2041 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2101 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2161 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2221 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2281 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     2341 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     2401 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     2461 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     2521 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     2581 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     2641 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     2701 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     2761 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2821 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2881 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2941 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3001 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3061 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3121 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3181 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3241 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3301 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3361 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3421 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3481 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3541 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3601 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3661 taaattccga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     3721 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     3781 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     3841 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     3901 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     3961 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     4021 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     4081 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     4141 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     4201 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
     4261 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     4321 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     4381 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     4441 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     4501 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
     4561 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     4621 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     4681 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     4741 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     4801 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact
     4861 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     4921 agcggataac aatttcacac aggaaacagc tatgaccatg attacggatt cactggccgt
     4981 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     5041 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     5101 acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg tttccggcac cagaagcggt
     5161 gccggaaagc tggctggagt gcgatcttcc tgaggccgat actgtcgtcg tcccctcaaa
     5221 ctggcagatg cacggttacg atgcgcccat ctacaccaac gtgacctatc ccattacggt
     5281 caatccgccg tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt
     5341 tgatgaaagc tggctacagg aaggccagac gcgaattatt tttgatggcg ttggaattag
     5401 c
//
