LOCUS       Exported                5648 bp ds-DNA     circular SYN 22-OCT-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5648)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Oct 22, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5648
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        183..211
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..1574
                     /codon_start=1
                     /label=StrepII-GPA1
                     /translation="MSASSAWSHPQFEKSGGSGTSGGSAWSHPQFEKVGSLVPRGSAGE
                     NLYFQGGSGSMGLLCSRSRHHTEDTDENTQAAEIERRIEQEAKAEKHIRKLLLLGAGES
                     GKSTIFKQIKLLFQTGFDEGELKSYVPVIHANVYQTIKLLHDGTKEFAQNETDSAKYML
                     SSESIAIGEKLSEIGGRLDYPRLTKDIAEGIETLWKDPAIQETCARGNELQVPDCTKYL
                     MENLKRLSDINYIPTKEDVLYARVRTTGVVEIQFSPVGENKKSGEVYRLFDVGGQRNER
                     RKWIHLFEGVTAVIFCAAISEYDQTLFEDEQKNRMMETKELFDWVLKQPCFEKTSFMLF
                     LNKFDIFEKKVLDVPLNVCEWFRDYQPVSSGKQEIEHAYEFVKKKFEELYYQNTAPDRV
                     DRVFKIYRTTALDQKLVKKTFKLVDETLRRRNLLEAGLL"
     CDS             258..260
                     /codon_start=1
                     /label=ATG
                     /translation="M"
     CDS             276..299
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             333..356
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             366..383
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             390..410
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             423..1574
                     /codon_start=1
                     /label=GPA1
                     /translation="MGLLCSRSRHHTEDTDENTQAAEIERRIEQEAKAEKHIRKLLLLG
                     AGESGKSTIFKQIKLLFQTGFDEGELKSYVPVIHANVYQTIKLLHDGTKEFAQNETDSA
                     KYMLSSESIAIGEKLSEIGGRLDYPRLTKDIAEGIETLWKDPAIQETCARGNELQVPDC
                     TKYLMENLKRLSDINYIPTKEDVLYARVRTTGVVEIQFSPVGENKKSGEVYRLFDVGGQ
                     RNERRKWIHLFEGVTAVIFCAAISEYDQTLFEDEQKNRMMETKELFDWVLKQPCFEKTS
                     FMLFLNKFDIFEKKVLDVPLNVCEWFRDYQPVSSGKQEIEHAYEFVKKKFEELYYQNTA
                     PDRVDRVFKIYRTTALDQKLVKKTFKLVDETLRRRNLLEAGLL"
     CDS             1596..1619
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        1951..2055
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2056..2916
                     /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"
     rep_origin      3087..3675
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        3919..3996
                     /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."
     CDS             3997..5079
                     /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"
     protein_bind    5092..5113
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5128..5158
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5166..5182
                     /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     5190..5206
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5202..5648
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGI"
     primer_bind     complement(5222..5238)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg agcgctagca gcgcgtggag ccacccgcag ttcgaaaaga
      301 gcggtggctc aggtaccagt ggcggttcag cctggagcca tccgcaattc gagaaggtcg
      361 gcagcctggt tccgcgtggt tctgctggtg aaaacctgta cttccagggt ggttcaggat
      421 ccatgggctt actctgcagt agaagtcgac atcatactga agatactgat gagaatacac
      481 aggctgctga aatcgaaaga cggatagagc aagaagcaaa ggctgaaaag catattcgga
      541 agcttttgct acttggtgct ggggaatctg gaaaatctac aatttttaag cagataaaac
      601 ttctattcca aacgggattt gatgaaggag aactaaagag ctatgttcca gtcattcatg
      661 ccaatgtcta tcagactata aaattattgc atgatggaac aaaggagttt gctcaaaatg
      721 aaacagattc tgctaaatat atgttatctt ctgaaagtat tgcaattggg gagaaactat
      781 ctgagattgg tggtaggtta gactatccac gtcttaccaa ggacatcgct gagggaatag
      841 aaacactatg gaaggatcct gcaattcagg aaacttgtgc tcgtggtaat gagcttcagg
      901 ttcctgattg tacgaaatat ctgatggaga acttgaagag actatcagat ataaattata
      961 ttccaactaa ggaggatgta ctttatgcaa gagttcgcac aactggtgtc gtggaaatac
     1021 agttcagccc tgtgggagag aataaaaaaa gtggtgaagt gtaccgattg tttgacgtgg
     1081 gtggacagag aaatgagagg aggaaatgga ttcatctgtt tgaaggtgta acagctgtga
     1141 tattttgtgc tgccatcagc gagtacgacc aaacgctctt tgaggacgag cagaaaaaca
     1201 ggatgatgga gaccaaggaa ttattcgact gggtcctgaa acaaccctgt tttgagaaaa
     1261 catccttcat gctgttcttg aacaagttcg acatatttga gaagaaagtt cttgacgttc
     1321 cgttgaacgt ttgcgagtgg ttcagagatt accaaccagt ttcaagtggg aaacaagaga
     1381 ttgagcatgc atacgagttt gtgaagaaga agtttgagga gttatattac cagaacacgg
     1441 cgccggatag agtggacagg gtattcaaaa tctacaggac gacggctttg gaccagaagc
     1501 ttgtaaagaa aacgttcaag ctcgtagatg agacactaag aaggagaaat ttactggagg
     1561 ctggcctttt atgatccgga gcgagcaccg gtgcagatta taaggatgat gatgataagt
     1621 aactgagtaa gcgatcgcag aattcatcgt gactgactga cgatctgcct cgcgcgtttc
     1681 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     1741 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     1801 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtataattc ttgaagacga
     1861 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     1921 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1981 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2041 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2101 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2161 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2221 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2281 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     2341 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2401 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2461 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2521 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2581 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2641 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2701 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2761 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2821 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2881 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2941 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3001 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3061 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3121 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3181 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3241 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3301 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3361 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3421 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3481 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3541 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3601 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3661 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3721 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3781 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3841 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3901 tcacaccgca taaattccga caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga
     3961 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
     4021 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
     4081 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
     4141 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
     4201 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
     4261 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
     4321 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
     4381 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
     4441 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
     4501 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
     4561 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
     4621 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
     4681 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
     4741 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc
     4801 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
     4861 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     4921 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     4981 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     5041 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     5101 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     5161 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacggatt
     5221 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     5281 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     5341 gcccttccca acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg tttccggcac
     5401 cagaagcggt gccggaaagc tggctggagt gcgatcttcc tgaggccgat actgtcgtcg
     5461 tcccctcaaa ctggcagatg cacggttacg atgcgcccat ctacaccaac gtaacctatc
     5521 ccattacggt caatccgccg tttgttccca cggagaatcc gacgggttgt tactcgctca
     5581 catttaatgt tgatgaaagc tggctacagg aaggccagac gcgaattatt tttgatggcg
     5641 ttggaatt
//
