LOCUS       sequence-333572-        6467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6467)
  AUTHORS   Lee HH, Kim DJ, Ahn HJ, Ha JY, Suh SW
  TITLE     Crystal structure of T-protein of the glycine cleavage system. 
            Cofactor binding, insights into H-protein recognition, and molecular
            basis for understanding nonketotic hyperglycinemia.
  JOURNAL   J Biol Chem. 2004 Nov 26. 279(48):50514-23.
  PUBMED    15355973
REFERENCE   2  (bases 1 to 6467)
  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: "J Biol 
            Chem. 2004 Nov 26. 279(48):50514-23."
COMMENT     Sequence Label: pET-21a(+)-T-protein
FEATURES             Location/Qualifiers
     source          1..6467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1199..1216
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1269..1287)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1283..1330
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1367..1822
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1454..1473)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1664..1685
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1848..1952
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1953..2021,2022..2813)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2171..2190)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2984..3572
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3473..3492
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3726..3743
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3758..3897
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3823..3842)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3983..4005
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3999..4190)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4999..6081)
                     /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     6043..6062
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6082..6159)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6365..6384
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gaagcgtacg cctctatttg aaaagcatgt
      121 tgaactcggt gcgaaaatgg tagatttcgc ggggtgggag atgcctcttt attacacatc
      181 catattcgaa gaagtgatgg cagtcagaaa atcggtgggg atgttcgatg tctctcacat
      241 gggggaattt ctcgttaaag ggcccgaggc ggtttctttt attgactttc ttataacgaa
      301 cgacttttct tcacttccgg atggtaaagc gatttactct gtcatgtgca atgaaaacgg
      361 tggaatcatc gatgatcttg ttgtgtacaa ggtgagtcct gatgaagctc tcatggtggt
      421 gaacgcggca aacattgaaa aggacttcaa ctggataaaa tcccattcga agaatttcga
      481 tgtcgaagta tcgaacattt cagatacgac agcgctcata gcgttccagg ggccaaaagc
      541 acaggagacc ctccaggagc ttgttgaaga cggtctcgag gaaattgcct attattcttt
      601 cagaaagagt attgtagccg gtgtagagac tcttgtctca agaacgggat acacgggtga
      661 ggacggtttc gaactgatgc ttgaagcgaa gaatgcaccg aaggtctggg atgctttgat
      721 gaatctgctc agaaaaatcg atggcagacc cgcaggtctt ggggcgagag acgtttgccg
      781 actcgaggcg acttatttgc tgtacggtca ggacatggat gagaacacga atcctttcga
      841 agtgggactt tcctgggttg taaaactgaa caaagacttc gtggggaagg aagcgctgct
      901 gaaagcgaag gaaaaagtgg aaagaaaact tgtggcgctt gagctctctg gaaaaagaat
      961 agcaagaaaa ggctatgaag ttttaaagaa tggagagaga gtgggagaga tcacaagtgg
     1021 taacttctct ccaactcttg ggaagtcgat agcgctggct ctcgtttcaa aatcggtgaa
     1081 gataggagac caactgggag tggtgtttcc tggtggaaag cttgtagaag ctctggtagt
     1141 aaaaaaacca ttttacagag gcagtgtgag gagggaggtt taagcggccg cactcgagca
     1201 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     1261 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     1321 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     1381 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1441 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1501 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1561 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1621 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1681 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1741 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     1801 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1861 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1921 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1981 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2041 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2101 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2161 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     2221 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2281 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2341 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2401 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2461 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     2521 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2581 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2641 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2701 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2761 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2821 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2881 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2941 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3001 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3061 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3121 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3181 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3241 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3301 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3361 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3421 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3481 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3541 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3601 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3661 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3721 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3781 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     3841 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3901 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3961 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4021 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     4081 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     4141 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     4201 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     4261 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4321 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4381 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4441 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     4501 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     4561 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4621 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     4681 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4741 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     4801 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     4861 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     4921 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4981 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5041 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     5101 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     5161 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     5221 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     5281 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     5341 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     5401 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     5461 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     5521 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     5581 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5641 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5701 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5761 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5821 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5881 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5941 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     6001 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     6061 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     6121 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     6181 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     6241 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     6301 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     6361 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     6421 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
