LOCUS       sequence-56997-e        6816 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 6816)
  AUTHORS   Lentini R, Forlin M, Martini L, Del Bianco C, Spencer AC, Torino D, 
            Mansy SS
  TITLE     Fluorescent Proteins and in Vitro Genetic Organization for Cell-Free
            Synthetic Biology.
  JOURNAL   ACS Synth Biol. 2013 Mar 15.
  PUBMED    23654270
REFERENCE   2  (bases 1 to 6816)
  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: "ACS Synth 
            Biol. 2013 Mar 15."
COMMENT     Sequence Label: pET21b-RL020A
FEATURES             Location/Qualifiers
     source          1..6816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        494..598
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(599..667,668..1459)
                     /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(817..836)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1630..2218
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2119..2138
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2372..2389
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2404..2546
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2472..2491)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2632..2654
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2648..2839)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3648..4730)
                     /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     4692..4711
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4731..4808)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5014..5033
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5117..5136
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5117..5135
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5136..5160
                     /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             5175..5197
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             5938..5960
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6685..6702
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6755..6773)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6769..6816
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
      601 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
      661 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
      721 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
      781 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      841 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      901 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      961 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     1021 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     1081 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     1141 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     1201 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     1261 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     1321 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     1381 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     1441 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     1501 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1561 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1621 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1681 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1741 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     1801 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1861 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1921 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1981 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     2041 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2101 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2161 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2221 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2281 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2341 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     2401 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg
     2461 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     2521 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     2581 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     2641 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     2701 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     2761 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     2821 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     2881 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     2941 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     3001 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     3061 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     3121 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     3181 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     3241 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     3301 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     3361 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     3421 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     3481 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     3541 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     3601 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     3661 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3721 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     3781 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     3841 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     3901 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     3961 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     4021 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     4081 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     4141 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     4201 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     4261 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     4321 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     4381 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     4441 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     4501 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     4561 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     4621 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     4681 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     4741 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     4801 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     4861 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     4921 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     4981 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     5041 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     5101 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     5161 cctctagaaa taattttgtt taactttaag aaggagatat acatatgagc aaaggcgaag
     5221 aactgttcac gggtgtggtt ccgatcctgg ttgaactgga tggcgatgtg aacggtcata
     5281 aatttagcgt gtctggtgaa ggcgaaggtg atgcgaccta cggcaaactg acgctgaaac
     5341 tgatttgcac cacgggtaaa ctgccggttc cgtggccgac cctggtgacc acgctgggtt
     5401 atggtctgat gtgtttcgca cgttacccgg atcacatgaa acgccatgat ttctttaaat
     5461 ctgcgatgcc ggaaggctat gtgcaggaac gtaccatctt tttcaaagat gatggtaact
     5521 acaaaacccg cgcggaagtt aaatttgaag gcgatacgct ggtgaaccgt attgaactga
     5581 aaggtatcga tttcaaagaa gatggcaata ttctgggtca caaactggaa tacaactaca
     5641 acagtcataa cgtgtacatt accgccgata aacagaaaaa cggtatcaaa gcaaacttca
     5701 aaatccgtca caacatcgaa gatggcggtg ttcagctggc cgatcattac cagcagaaca
     5761 ccccgattgg cgatggtccg gtgctgctgc cggataatca ttatctgagt taccagagca
     5821 aactgtctaa agatccgaat gaaaaacgcg atcacatggt tctgctggaa tttgtgaccg
     5881 cggccggcat tacgcatggt atggatgaac tgtataaata agcggatcct gaataatttt
     5941 gtttaacttt aagaaggaga tatacatatg gtgagtaaag gcgaggagga caatatggcg
     6001 atcatcaaag agttcatgcg cttcaaagtc cacatggaag gcagcgttaa tggtcacgag
     6061 ttcgaaattg agggcgaagg cgaaggtcgt ccgtatgagg gtacacagac cgctaaactg
     6121 aaagtcacga aaggtggtcc actgccattt gcttgggata ttctgagccc acagttcatg
     6181 tatggctcca aagcctatgt gaaacatccg gccgatattc cggactatct gaaactgagc
     6241 ttccctgaag ggttcaaatg ggaacgtgtg atgaactttg aggatggtgg tgttgtgaca
     6301 gtgacacagg attctagcct gcaagacggt gagttcatct ataaagtgaa actgcgtggc
     6361 acgaattttc cgagtgatgg cccggttatg cagaaaaaaa cgatgggttg ggaggcctct
     6421 agtgagcgta tgtatccaga agatggcgct ctgaaaggcg aaatcaaaca gcgtctgaaa
     6481 ctgaaagatg gtggccacta tgatgccgaa gtgaaaacca cgtataaagc caaaaaacct
     6541 gtccaactgc ctggtgccta taacgttaac atcaaactgg acatcacctc acacaatgag
     6601 gactatacga tcgtggagca gtatgagcgt gctgaaggac gtcattctac cggtggtatg
     6661 gatgagctgt ataaataact cgagcaccac caccaccacc actgagatcc ggctgctaac
     6721 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     6781 cttggggcct ctaaacgggt cttgaggggt tttttg
//
